Muhammad A Farooq

Sébastien Gaertner

Lamia Amoura

Zahid R Niazi

Sin-Hee Park

Abdul W Qureshi

Min-Ho Oak

Florence Toti

Valérie B Schini-Kerth

PhD Cyril Auger
email: cyril.auger@unistra.fr

Sin- Hee Park

Intake of omega-3 formulation EPA:DHA 6:1 by old rats for 2 weeks improved endothelium-dependent relaxations and normalized the expression level of ACE/AT1R/NADPH oxidase and the formation of ROS in the mesenteric artery

Keywords: (6 max): Aging, Omega-3 polyunsaturated fatty acids, Artery, Endothelium, Nitric oxide, Angiotensin system

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Introduction

Cardiovascular diseases such as ischemic heart disease and stroke are a major cause of death responsible for 32% of deaths worldwide in 2013 [START_REF] Roth | Global and regional patterns in cardiovascular mortality from 1990 to 2013[END_REF]. Age has been identified as a main risk factor for the development of cardiovascular diseases. The impact of aging on the vascular system is characterized predominantly by increased thickness and stiffness of the arterial wall and impaired regulation of vascular tone as indicated by the appearance of endothelial dysfunction [START_REF] North | The intersection between aging and cardiovascular disease[END_REF][START_REF] Lakatta | Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a "set up" for vascular disease[END_REF]. Under physiological conditions, the vascular endothelium has a pivotal protective role in the control of vascular homeostasis mainly by inducing potent vasoprotective mechanisms including the formation of endothelium-derived nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH). Both these responses are blunted with increasing age in experimental animals and humans and often are associated with the appearance of endothelium-dependent contractile responses (EDCFs) [START_REF] Dal-Ros | Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress[END_REF][START_REF] Assar | Oxidative stress and vascular inflammation in aging[END_REF][START_REF] Idris Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF][START_REF] Taddei | Aging and endothelial function in normotensive subjects and patients with essential hypertension[END_REF]. Moreover, old arteries are characterized by increased formation of reactive oxygen species (ROS) throughout the arterial wall involving several cellular sources such as NADPH oxidase, cyclooxygenases (COXs), the mitochondrial respiratory chain, and uncoupled endothelial NOS (eNOS) [START_REF] Dal-Ros | Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress[END_REF][START_REF] Idris Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF].

Previous studies have shown a pivotal role of both circulating and local angiotensin systems in the induction of endothelial dysfunction in several experimental models of hypertension, atherosclerosis, diabetes, and aging [START_REF] Dal-Ros | Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress[END_REF][START_REF] Idris Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF][START_REF] Dal-Ros | Angiotensin II-induced hypertension is associated with a selective inhibition of endotheliumderived hyperpolarizing factor-mediated responses in the rat mesenteric artery[END_REF][START_REF] Kane | Endothelium-derived contracting factors mediate the Ang IIinduced endothelial dysfunction in the rat aorta: preventive effect of red wine polyphenols[END_REF][START_REF] Lee | An ethanolic extract of Lindera obtusiloba stems, YJP-14, improves endothelial dysfunction, metabolic parameters and physical performance in diabetic db/db mice[END_REF] and also in humans [START_REF] Mehta | Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system[END_REF]. Angiotensin II (Ang II)-induced endothelial dysfunction is characterized by the vascular formation of ROS subsequent to the upregulation of the expression level of NADPH oxidase [START_REF] Harrison | Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease[END_REF][START_REF] Sunggip | Redox control of cardiovascular homeostasis by angiotensin II[END_REF], thereby promoting eNOS uncoupling to further generate ROS [START_REF] Lee | Nox4 NADPH Oxidase Mediates Peroxynitrite-dependent Uncoupling of Endothelial Nitric-oxide Synthase and Fibronectin Expression in Response to Angiotensin II: ROLE OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES[END_REF], alteration of calcium-dependent K + channels involved in EDH responses [START_REF] Behringer | Aging impairs electrical conduction along endothelium of resistance arteries through enhanced Ca2+-activated K+ channel activation[END_REF], and increased expression of COXs, promoting EDCF responses [START_REF] Feletou | Endothelium-mediated control of vascular tone: COX-1 and COX-2 products[END_REF][START_REF] Ohnaka | Induction of cyclooxygenase-2 by angiotensin II in cultured rat vascular smooth muscle cells[END_REF].

Recently, premature endothelial senescence has been suggested to be an early event contributing to age-related endothelial dysfunction and the development of cardiovascular diseases [START_REF] Herrera | Endothelial dysfunction and aging: an update[END_REF]. Premature endothelial senescence is observed in response to several pro-oxidant stimuli such as H2O2, Ang II, and elevated glucose concentration [START_REF] Khemais-Benkhiat | The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract[END_REF][START_REF] Erusalimsky | Vascular endothelial senescence: from mechanisms to pathophysiology[END_REF]. These prosenescence inducers promote the downregulation of eNOS and the induction of a proinflammatory and pro-atherothrombotic phenotype in endothelial cells [START_REF] Botden | Red wine extract protects against oxidative-stress-induced endothelial senescence[END_REF][START_REF] Silva | Replicative senescence promotes prothrombotic responses in endothelial cells: Role of NADPH oxidase-and cyclooxygenase-derived oxidative stress[END_REF][START_REF] Khemais-Benkhiat | Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence[END_REF].

Several, but not all clinical studies, have indicated that omega-3 polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), improve the endothelial function and reduce cardiovascular events in patients with cardiovascular risk factors [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Maki | Use of supplemental long-chain omega-3 fatty acids and risk for cardiac death: An updated meta-analysis and review of research gaps[END_REF]. Recently, the REDUCE-IT clinical trial has reported a decreased risk of cardiovascular events after supplementation with 2 g of an EPA ethyl ester (icosapent ethyl) twice daily in patients with established cardiovascular disease or with diabetes and other risk factors, who were on statin therapy and had a fasting triglyceride level of 135 to 499 mg/dL [START_REF] Bhatt | REDUCE-IT Investigators, Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia[END_REF]. The beneficial effect was stronger in patients with elevated baseline triglycerides despite moderate-to-high statin therapy intensity [START_REF] Bhatt | REDUCE-IT Investigators, Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia[END_REF] and observed across a wide range of baseline triglycerides [START_REF] Bhatt | Reduction in First and Total Ischemic Events With Icosapent Ethyl Across Baseline Triglyceride Tertiles[END_REF]. A beneficial effect on the cardiovascular system is also supported by the fact that both purified EPA and DHA alone induced endothelium-dependent relaxations of isolated rat conductance and resistance arteries mainly through stimulation of both NO and EDH pathways [START_REF] Limbu | Characterisation of the vasodilation effects of DHA and EPA, n-3 PUFAs (fish oils), in rat aorta and mesenteric resistance arteries[END_REF]. Our previous study has indicated that both the ratio and the purity of the EPA:DHA formulations are of major importance for the biological activity, as highly purified EPA:DHA 6:1 and 9:1 formulations demonstrated greater endothelium-dependent NO-mediated relaxation of porcine coronary artery rings than other ratios [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1[END_REF] and acted through a redox-sensitive activation of Src/PI3-kinase/Akt and MAPK pathways, leading to eNOS phosphorylation at Ser1177 and subsequent formation of NO [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1[END_REF]. EPA:DHA 6:1 also induced endothelium-dependent NO-mediated relaxations and inhibited serotonin-induced contractile responses in human internal thoracic artery rings [START_REF] Zgheel | EPA:DHA 6:1 is a superior omega-3 PUFAs formulation attenuating platelets-induced contractile responses in porcine coronary and human internal mammary artery by targeting the serotonin pathway via an increased endothelial formation of nitric oxide[END_REF].

Moreover, oral intake of EPA:DHA 6:1 significantly prevented Ang II-induced hypertension, endothelial dysfunction, and vascular pro-oxidant responses in rats [START_REF] Niazi | EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase-and COX-derived oxidative stress[END_REF].

The aim of the study was to evaluate the potential of a 2-week oral intake of EPA:DHA 6:1 to improve an established blunted endothelium-dependent relaxation in old rats and, if so, to determine the underlying mechanism and, in particular, the role of the pro-oxidant and the pro-senescence angiotensin system.

Methods and materials

Ethics statement

Chemicals

All chemicals were purchased from Sigma-Aldrich (Sigma Aldrich Chimie S.a.r.l, St.-Quentin-Fallavier, France) except TRAM-34, UCL-1684, levcromakalim, NS-398, and SC-560, which were bought from Tocris (Bio-Techne, Abingdon, UK), VAS-2870 from Enzo Life Science (Villeurbanne, France), and PBS and Triton X100 from Euromedex (Souffelweyersheim, France).

Preparation of Omega-3 PUFAs

Highly purified EPA and DHA products were provided by Pivotal Therapeutics, Inc.

(Woodbridge, ON, Canada). EPA:DHA 6:1 (w/w) was prepared by mixing the respective purified products in the ratio. The final solution was aliquoted in amber-colored vials. All the processes were done under nitrogen flux to avoid oxidation of omega-3 PUFAs.

In vivo treatment of rats

Thirty male Wistar rats were kept in the animal facility and given free access to standard diet and tap water from the age of 12 weeks until they were 20 months old. They were then divided randomly into 3 groups and administered daily with 500 mg/kg/day of either EPA:DHA 6:1, corn oil (isocaloric control without omega-3 PUFAs, Mazola 100% corn oil), or untreated (control old rats) for 14 days. A group of untreated 12-week-old rats was used as young controls. After 14 days of treatment, the rats were euthanized by an intraperitoneal injection of a lethal dose of pentobarbital (150 mg/kg) before collection of organs.

Vascular reactivity study

The main mesenteric artery was dissected, cleaned of connective tissue, and cut into rings (2-3 mm) before suspension in organ baths containing oxygenated (95% O2, 5% CO2) Krebs bicarbonate solution (composition in mM: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.18, CaCl2 1.25, NaHCO3 25, and D-glucose 11, pH 7.4, 37 °C) for the determination of changes in isometric tension. The rings were allowed to stabilize for 30-45 min at a tension of 1 g before the reactivity of the vascular smooth muscle was assessed in response to Krebs buffer containing 80 mM potassium. The integrity of the endothelium was checked with acetylcholine (ACh 1 µM) after sub-maximal contraction (approximately 70%) with phenylephrine (PE 1 µM). To assess the endothelium-dependent relaxations, the rings were contracted with PE (1 µM) before the construction of concentration-relaxation curves in response to ACh. To assess the contractile responses, concentration-contraction curves were constructed in response to PE. In some experiments, the rings were exposed to a pharmacological agent for 30 min before contraction. To study the role of cyclooxygenasederived prostanoids, the rings were incubated with indomethacin (10 µM, a nonselective COX inhibitor). To study the role of NO-mediated relaxation, the rings were incubated in the presence of indomethacin and TRAM-34 plus UCL-1684 (1 µM each, inhibitors of IKCa and SKCa, respectively) to prevent the formation of vasoactive prostanoids and EDH-mediated relaxation, respectively. The role of EDH-mediated relaxation was studied in rings incubated with indomethacin and N ω -nitro-L-arginine (L-NA, 300 µM, an eNOS inhibitor) to prevent the formation of vasoactive prostanoids and NO, respectively. To study endotheliumdependent vasoconstrictor factors (EDCFs), the rings were exposed to L-NA and TRAM-34 plus UCL-1684 to prevent NO and EDH, respectively, in the absence or presence of indomethacin, before an approximate 30% pre-contraction with PE and the subsequent construction of a concentration-contraction curve in response to ACh. To assess the vascular smooth muscle function, the rings were contracted with PE (1 µM) before the construction of concentration-relaxation curves in response to either sodium nitroprusside (SNP, a NO donor) or levcromakalim (Lev, an ATP-sensitive potassium channel opener) in the presence of indomethacin, L-NA, and TRAM-34 plus UCL-1684.

Immunofluorescence studies

Rings of the main mesenteric artery and thoracic aorta were embedded in histomolds containing Tissue-Tek optimum cutting temperature (OCT) compound (Sakura 4583, Leiden, The Netherlands) and snap-frozen in liquid nitrogen. Rings were cryosectioned at 14 μm and stored at -80 °C until use. The sections were defrosted with phosphate-buffered saline (PBS) and fixed for 1 h with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) before blocking for 2 h at room temperature with 10% non-fat powdered milk in PBS containing 0.1% Triton X-100 to prevent nonspecific binding. All sections, excluding negative controls, were then incubated overnight at 4 °C with a solution of blocking buffer containing a primary antibody against either COX-1 (1/250, Cat. ab109025, Abcam, Paris, USA) and dried for 20 min at room temperature. The slides were then analyzed using a confocal laser scanning microscope (Leica TSC SPE, Mannheim, Germany). Signals were collected in the red wavelength between 645 and 719 nm following excitation of Alexa 633 using the 635 nm red laser. Autofluorescence was measured in the green wavelength between 500 and 600 nm following excitation with the 488 nm cyan laser. Quantitative analysis of fluorescence was performed using ImageJ software (version 1.49 for Windows, NIH). The mean fluorescence intensity of the elastic lamina was measured in both green and red channels to calculate the signal ratio between the two channels. The autofluorescence signal (green channel) is then subtracted for the signal of the red channel using the signal ratio. The mean fluorescence signal in the resulting image is then determined in the area of interest delimited manually. The mean fluorescence signal was measured in the endothelial layer for eNOS and in the endothelium plus media for the other proteins.

Determination of the in situ level of ROS

The vascular level of ROS was determined using the redox-sensitive fluorescent probe dihydroethidium (DHE, Calbiochem, Sigma-Aldrich). Cryosections of the mesenteric artery or the aorta (25 µm) were defrosted with PBS and incubated with DHE (2.5 µM) for 30 min at 37 °C in a black box to protect them from light. To determine the sources of ROS, aorta sections were pretreated with inhibitors such as N-acetylcysteine (antioxidant), indomethacin (inhibitor of COX), NS-398 (COX-2 inhibitor), SC-560 (COX-1 inhibitor), L-NA (inhibitor of eNOS), VAS-2870 (inhibitor of NADPH oxidase), and inhibitors of mitochondrial respiratory chain (KCN, myxothiazol, potassium cyanide, and rotenone) for 30 min at 37 °C before incubation with DHE. The sections were then washed with PBS before mounting under a cover slip in DAKO fluorescence solution. The slides were allowed to dry in the dark for 20 min before being analyzed by confocal laser scanning microscopy. The images were processed as in section 2.6 except that signals were collected between 580 and 650 nm following the excitation of the ethidium with the 561 nm yellow-green laser, and autofluorescence between 495 and 581 nm following excitation with the 488 nm cyan laser.

Statistical analysis

Values were expressed as mean ± S.E.M. Statistical analysis was performed by two-way analysis of variance (ANOVA), followed by Bonferroni's post-hoc test for vascular reactivity and by two-tailed unpaired t-test for other data. The normality of the data was verified by Shapiro-Wilk test. Values were considered to show statistically significant difference for P<0.05. All statistical analyses were performed using GraphPad Prism software (version 5 for Windows, GraphPad Software, Inc., San Diego, CA, USA).

Results

Intake of EPA:DHA 6:1 by old rats for 2 weeks improves vascular reactivity of intact mesenteric artery rings

To assess the vascular effect of a 2-week oral intake of the omega-3 EPA:DHA 6:1 formulation by old rats, the reactivity of the main mesenteric artery with endothelium was studied using organ chambers. After precontraction with phenylephrine (PE, 1 µM), the rings from old rats showed significantly decreased relaxations in response to acetylcholine (ACh, 0.1-10 µM) when compared with the rings from young rats (Figure 1A). Similarly, the rings from old rats showed significantly increased contractile responses to increasing concentrations of PE (1-10 µM) when compared with those from young rats (Figure 1B).

Treatment with EPA:DHA 6:1, but not with corn oil, significantly improved ACh-induced relaxations and reduced PE-induced contractions in the mesenteric artery rings of old rats (Figures 1A,B).

To assess EDCF-mediated responses, the rings were preincubated with N ω -nitro-L-arginine (L-NA, an eNOS inhibitor) and TRAM-34 plus UCL-1684 (inhibitors of IKCa and SKCa, respectively) to prevent NO-and EDH-mediated relaxation, respectively. Rings from untreated old rats showed significantly greater contractions in response to ACh than those of young rats (Figure 1C), such responses were abolished in the presence of indomethacin (a nonselective COX inhibitor, Figure 1D), indicating the involvement of an increased formation of COX-derived EDCFs. Treatment of old rats with EPA:DHA 6:1 partially, but significantly, reduced EDCF-mediated contractile responses in the mesenteric artery, whereas corn oil was without effect (Figure 1C).

In addition, the endothelium-independent relaxations to either SNP (an NO donor) or levcromakalim (Lev, an ATP-sensitive potassium channel opener) were similar in old and young rats, indicating that the function of the vascular smooth muscle, in contrast to endothelial function, is preserved in the old mesenteric artery (Figures 1E,F).

EPA:DHA 6:1 treatment improves the NO-mediated relaxation and decreases the PE-induced contractile response in the old mesenteric artery

In the mesenteric artery rings of young rats, the ACh-induced relaxation was significantly reduced in the presence of L-NA and slightly but significantly in the presence of TRAM-34 plus UCL-1684 (from 50.4±5.3% to 25.6±7.1% at 3.10 -8 M of ACh for control and in the presence of TRAM-34 plus UCL-1684, respectively), indicating the involvement predominantly of NO and, to some extent, of EDH (Figure 2A, Table 1). In contrast to young rats, the ACh-induced relaxation in the rings from control old rats was not affected by TRAM-34 plus UCL-1684 but abolished in the presence of L-NA, indicating that the relaxation is exclusively mediated by NO (Figure 2B, Table 1). In addition, indomethacin slightly improved the relaxation to ACh at concentrations greater than 1 µM in old rats, but this effect did not reach statistical significance (Table 1).

Similarly, in old rats treated with either EPA:DHA 6:1 or corn oil, the relaxation to ACh was not affected by TRAM-34 plus UCL-1684 and by indomethacin but abolished in the presence of L-NA, indicating that they are exclusively mediated by NO (Figures 2 C,D,Table 1).

In the mesenteric artery rings of old but not young rats, the PE-induced contractile response was significantly reduced in the presence of indomethacin, suggesting the involvement of COX-derived vasoconstrictor prostanoids (Figure 2F, Table 1). The inhibitory effect of indomethacin was also observed in the corn oil-treated group but not in the EPA:DHA 6:1treated group of old rats. In addition, the contractile response to PE was markedly increased in the presence of L-NA in the EPA:DHA-treated group but not in the control and corn oiltreated groups of old rats, suggesting an increased basal formation on NO (Figure 2 G,H, Table 1).

EPA:DHA 6:1 treatment normalizes the expression level of eNOS and COXs in the old mesenteric artery and aorta

As the endothelial dysfunction of the old mesenteric artery is characterized by a reduced NOmediated relaxation and increased COX-dependent EDCF-mediated contractile response, the expression level of proteins involved in these pathways, eNOS and COXs, respectively, was assessed by immunofluorescence in both the mesenteric artery and the aorta. The eNOS fluorescent signal was observed selectively in the endothelium, whereas that of COX-1 and COX-2 was observed throughout the arterial wall in the mesenteric artery and the aorta in all groups (Figure 3). Compared to young rats, sections of old arteries showed higher eNOS and COX-2 signals but lower COX-1 signals (Figure 3). The EPA:DHA 6:1 but not the corn oil treatment normalized eNOS, COX-1, and COX-2 signals in old arteries to a similar level as those observed in young arteries (Figure 3).

EPA:DHA 6:1 treatment normalizes the formation of ROS in the old mesenteric artery and aorta

As age-related endothelial dysfunction is associated with an increased level of oxidative stress [START_REF] Dal-Ros | Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress[END_REF][START_REF] Assar | Oxidative stress and vascular inflammation in aging[END_REF][START_REF] Idris Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF], the formation of ROS was assessed in the mesenteric artery and aorta using the redoxsensitive probe DHE. Sections of the mesenteric artery and aorta of control and corn oiltreated old rats showed similar higher levels of ethidium fluorescence as those of young rats, demonstrating an increased formation of ROS (Figures 4 A,B). The EPA:DHA 6:1 but not the corn oil treatment normalized the level of ROS in both the old mesenteric artery and aorta to a similar level as that observed in young arteries (Figures 4 A,B).

In addition, the formation of ROS in the aortic section of control old rats was significantly lower in the presence of N-acetylcysteine (NAC, an antioxidant), indomethacin (nonselective COX inhibitor), SC-560 (a selective COX-1 inhibitor), NS-398 (a selective COX-2 inhibitor), VAS-2870 (a NADPH oxidase inhibitor), L-NA (eNOS inhibitor) or by the combination of potassium cyanide, rotenone, and myxothiazol (inhibitors of the mitochondrial respiratory chain, Figure 4C), indicating the involvement of several sources of ROS including COX-1 and COX-2, uncoupled eNOS, NADPH oxidase, and mitochondrial respiratory chain (Figure 4C).

EPA:DHA 6:1 treatment improves the expression level of p47 phox and p22 phox

NADPH oxidase subunits in the old aorta

As the age-related increased ROS formation involves NADPH oxidase, a major cellular source of ROS in cardiovascular diseases [START_REF] Lassegue | NADPH oxidases: functions and pathologies in the vasculature[END_REF], the expression level of the NADPH oxidase subunits p47 phox and p22 phox was assessed in the aorta by immunofluorescence. Compared to young rats, the fluorescent signals of both p47 phox and p22 phox NADPH oxidase subunits were significantly higher in the old aorta (Figure 5). The EPA:DHA 6:1 but not the corn oil treatment normalized the signals of both NADPH oxidase subunits in the old aorta (Figure 5).

EPA:DHA 6:1 treatment improves the expression level of ACE and AT1R in the old aorta

As an angiotensin-converting enzyme (ACE) inhibitor and an AT1R antagonist improved agerelated endothelial dysfunction and excessive vascular ROS formation [START_REF] Idris Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF][START_REF] Mukai | Inhibition of renin-angiotensin system ameliorates endothelial dysfunction associated with aging in rats[END_REF], the expression level of two major components of the local angiotensin system, namely, ACE and angiotensin II type 1 receptor (AT1R), was assessed by immunofluorescence. Aortic sections of control old rats showed higher fluorescence signals of both ACE and AT1R than those of young rats (Figure 6).The EPA:DHA 6:1 but not corn oil treatment normalized signals of both ACE and AT1R in the old aorta to a similar level as those observed in the young aorta (Figure 6).

EPA:DHA 6:1 treatment improves the expression level of the senescence markers p53, p21, and p16 in the old aorta

As endothelial senescence has been identified as an early event leading to endothelial dysfunction [START_REF] Kim | p53 impairs endothelium-dependent vasomotor function through transcriptional upregulation of p66shc[END_REF], the expression level of senescence markers was assessed by immunofluorescence. Aortic sections of old rats showed a higher fluorescence signal of p53, p21, and p16 throughout the arterial wall than those of young rats (Figure 7). EPA:DHA 6:1 but not corn oil treatment normalized signals of p53, p21, and p16 in the old aorta to a similar level as those observed in the young aorta (Figure 7).

Discussion

Both preclinical and clinical studies have indicated that the progressive increase in age is associated with the development of endothelial dysfunction, as observed in the present study in the mesenteric artery of old rats. The characterization of age-related endothelial dysfunction has indicated the involvement of a reduced NO-mediated relaxation, induction of EDCF-mediated contractile responses, and induction of vascular senescence as pointed out by an upregulation of the senescence markers p53, p21, and p16. Old mesenteric arteries are also characterized by an increased ROS formation involving several sources and, in particular, NADPH oxidase. As old arteries showed upregulation of the expression level of ACE and ATR1, whose activation promotes NAPDH oxidase-derived formation of ROS, the local angiotensin system seems to be involved. Moreover, the present findings also show that intake of EPA:DHA 6:1 by old rats for 2 weeks improved the age-related endothelial dysfunction, normalized the vascular ROS formation, and normalized the expression of ACE and AT1R and senescence markers.

Circulating levels of omega-3 PUFAs have been inversely associated with the risk of cardiovascular diseases. Indeed, a low content of omega-3 PUFAs in erythrocyte membranes has been associated with an increased risk of cardiovascular and all-cause mortality in the Framingham Heart Study Offspring cohort [START_REF] Harris | Erythrocyte long-chain omega-3 fatty acid levels are inversely associated with mortality and with incident cardiovascular disease: The Framingham Heart Study[END_REF]. Several clinical studies and meta-analyses have evaluated the effects of various omega-3 PUFAs-rich products (fish, fish oil, marine oil, krill oil, etc.) or purified omega-3 PUFAs such as EPA and DHA in primary and secondary cardiovascular outcome trials [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Dinicolantonio | Omega-3s and cardiovascular health[END_REF][START_REF] Bhatt | REDUCE-IT Investigators, Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia[END_REF][START_REF] Bhatt | Reduction in First and Total Ischemic Events With Icosapent Ethyl Across Baseline Triglyceride Tertiles[END_REF][START_REF] Iwamatsu | Which has the stronger impact on coronary artery disease, eicosapentaenoic acid or docosahexaenoic acid?[END_REF][START_REF] Aung | Associations of Omega-3 Fatty Acid Supplement Use With Cardiovascular Disease Risks: Metaanalysis of 10 Trials Involving 77 917 Individuals[END_REF]. Although some studies reported a beneficial effect of omega-3 PUFA supplementation on major cardiovascular endpoints [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Maki | Use of supplemental long-chain omega-3 fatty acids and risk for cardiac death: An updated meta-analysis and review of research gaps[END_REF][START_REF]Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial[END_REF], no such effect was observed in other studies and meta-analyses [START_REF] Aung | Associations of Omega-3 Fatty Acid Supplement Use With Cardiovascular Disease Risks: Metaanalysis of 10 Trials Involving 77 917 Individuals[END_REF][START_REF] Enns | The impact of omega-3 polyunsaturated fatty acid supplementation on the incidence of cardiovascular events and complications in peripheral arterial disease: a systematic review and meta-analysis[END_REF]. Such differences in outcomes might possibly relate to differences in study design, heterogeneity in the patient population and in the endpoints reported, the fact that patients in the more recent trials were optimally treated with statins, and most likely the use of different doses, omega-3 PUFA sources, the degree of purity, and the formulation of the omega-3 PUFA products [START_REF] Kromhout | Omega-3 fatty acids and coronary heart disease. The final verdict?[END_REF].

The dose of 500 mg/kg/day of EPA:DHA 6:1 used in the present study is equivalent to 5.67 g/day of omega-3 PUFAs in a 70 kg human [START_REF] Reagan-Shaw | Dose translation from animal to human studies revisited[END_REF], which is within the range of doses reported in different clinical studies, ranging from 0.18 to up to 10 g/day [START_REF] Delgado-Lista | Long chain omega-3 fatty acids and cardiovascular disease: a systematic review[END_REF][START_REF] Bhatt | REDUCE-IT Investigators, Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia[END_REF][START_REF] Enns | The impact of omega-3 polyunsaturated fatty acid supplementation on the incidence of cardiovascular events and complications in peripheral arterial disease: a systematic review and meta-analysis[END_REF][START_REF] Appel | Does supplementation of diet with "fish oil" reduce blood pressure? A meta-analysis of controlled clinical trials[END_REF][START_REF] Miller | Long-chain omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and blood pressure: a meta-analysis of randomized controlled trials[END_REF]. In a previous preclinical study with hypertensive rats, we have shown that oral intake of 500 mg/kg/day of the EPA:DHA 6:1 formulation was associated with increased circulating levels of omega-3 PUFAs and improved Omega Score and n-6/n-3 ratio [START_REF] Niazi | EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase-and COX-derived oxidative stress[END_REF]. The beneficial effects of omega-3 PUFAs on the cardiovascular system have been attributable to several effects such as reduction of the chronic inflammatory response, inhibition of the thrombogenesis, reduction of hypertension, and improvement of the myocardial function [START_REF] Calvo | Omega-3 polyunsaturated fatty acids and cardiovascular health: a molecular view into structure and function[END_REF].

Previous studies have indicated that the age-related endothelial dysfunction is characterized by blunted NO-and often also EDH-components of the endothelium-dependent relaxation as observed in the mesenteric artery [4,6, present findings], femoral arteries [START_REF] Puzserova | Age-related alterations in endothelial function of femoral artery in young SHR and WKY rats[END_REF], aorta [START_REF] Van Der Loo | Enhanced peroxynitrite formation is associated with vascular aging[END_REF], and coronary arterioles [START_REF] Csiszar | Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function[END_REF] of rats. The present study indicates that in the old mesenteric artery, the EDH component is abolished, whereas the NO component is partially but significantly decreased. Moreover, a 2-week oral intake of EPA:DHA 6:1 by old rats significantly improved NO-mediated relaxations but not the EDH component. The fact that eNOS inhibition by L-NA increased to a greater extent the PE-induced contractions in the EPA:DHA 6:1-treated group than in the control group of old rats suggests an increased basal release of NO. This is in agreement with our previous investigations showing that EPA:DHA 6:1 is a potent stimulator of the endothelial formation of NO in isolated porcine coronary arteries [START_REF] Zgheel | Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1[END_REF] and human internal thoracic artery [START_REF] Zgheel | EPA:DHA 6:1 is a superior omega-3 PUFAs formulation attenuating platelets-induced contractile responses in porcine coronary and human internal mammary artery by targeting the serotonin pathway via an increased endothelial formation of nitric oxide[END_REF].

The local vascular angiotensin system appears to have a major role in the induction of endothelial dysfunction and the associated vascular oxidative stress in in vitro and preclinical models of aging and hypertension [START_REF] Dal-Ros | Angiotensin II-induced hypertension is associated with a selective inhibition of endotheliumderived hyperpolarizing factor-mediated responses in the rat mesenteric artery[END_REF][START_REF] Harrison | Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease[END_REF][START_REF] Rajagopalan | Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone[END_REF]. Indeed, ACE inhibitors and/or AT1R antagonists restored, at least partially, the endothelial function in old rats and normalized the level of vascular oxidative stress [START_REF] Mukai | Inhibition of renin-angiotensin system ameliorates endothelial dysfunction associated with aging in rats[END_REF][START_REF] Goto | Angiotensin-converting enzyme inhibitor prevents age-related endothelial dysfunction[END_REF][START_REF] Kansui | Angiotensin II receptor antagonist improves agerelated endothelial dysfunction[END_REF], indicating the involvement of the AT1R-mediated activation of NADPH oxidase [START_REF] Harrison | Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease[END_REF][START_REF] Rajagopalan | Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone[END_REF]. In the present study, the increased expression level of ACE and AT1R points to an increased activation of the local angiotensin system as observed previously in old rats [START_REF] Yoon | Age-Associated Changes in the Vascular Renin-Angiotensin System in Mice[END_REF]. This is also consistent with the observation that the NADPH oxidase inhibitor VAS-2870 reduced the level of ethidium fluorescence and that an upregulation of the expression of the NADPH oxidase subunits p22 phox and p47 phox was observed in the old artery. The EPA:DHA 6:1 treatment of old rats normalized the activation of the local angiotensin system including ACE, AT1R, NADPH oxidase subunits p22 phox and p47 phox , and formation of ROS to a level similar to that observed in young rats. Similar observations to the EPA:DHA 6:1 treatment have been observed in Ang II-induced hypertensive rats [START_REF] Niazi | EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase-and COX-derived oxidative stress[END_REF].

Vascular oxidative stress has also been shown to contribute to age-related vascular alterations [START_REF] Ungvari | Oxidative stress in vascular senescence: lessons from successfully aging species[END_REF] and endothelial dysfunction in the mesenteric artery of middle-aged rats [START_REF] Dal-Ros | Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress[END_REF][START_REF] Idris Khodja | Grape-derived polyphenols improve aging-related endothelial dysfunction in rat mesenteric artery: role of oxidative stress and the angiotensin system[END_REF]. The agerelated increased formation of ROS involving several vascular sources including COXs, uncoupled eNOS, NADPH oxidase, and the mitochondrial respiratory chain can cause direct inactivation of NO by chemically converting it to peroxynitrite anion (ONOO •-) and a reduced formation of NO subsequent to the oxidation of BH4, the most important cofactor of eNOS, thus leading to uncoupled eNOS to further promote the formation of ROS [START_REF] Yang | Sun, eNOS uncoupling and endothelial dysfunction in aged vessels[END_REF]. Uncoupling of eNOS is strongly suggested in the present study by the fact that the increased formation of ROS in the old artery is reduced in the presence of the eNOS inhibitor L-NA. Moreover, the increased expression level of eNOS in the old artery is mostly likely part of a compensatory mechanism subsequent to the reduced bioavailability of NO.

Previous studies have reported that premature endothelial senescence leads to endothelial dysfunction [START_REF] Herrera | Endothelial dysfunction and aging: an update[END_REF][START_REF] Jane-Wit | Mechanisms of dysfunction in senescent pulmonary endothelium[END_REF]. Several cellular stresses including oxidative stress, angiotensin II, high glucose, and reduced availability of NO are associated with the induction of premature endothelial senescence, which causes the upregulation of the cyclin-dependent kinase inhibitors p53, p21, and p16, leading to cell cycle arrest [START_REF] Erusalimsky | Vascular endothelial senescence: from mechanisms to pathophysiology[END_REF]. Moreover, replicative endothelial senescence is associated with an upregulation of the angiotensin system (ACE, AT1R) and NADPH oxidase and increased oxidative stress [START_REF] Khemais-Benkhiat | The Redox-sensitive Induction of the Local Angiotensin System Promotes Both Premature and Replicative Endothelial Senescence: Preventive Effect of a Standardized Crataegus Extract[END_REF][START_REF] Silva | Replicative senescence promotes prothrombotic responses in endothelial cells: Role of NADPH oxidase-and cyclooxygenase-derived oxidative stress[END_REF]. The present study shows an upregulation of p53, p21, and p16 in the old aorta, suggesting the induction of vascular senescence, which is normalized by the EPA:DHA 6:1 treatment. A previously published study has also shown that omega-3 PUFAs prevented hydrogen peroxide-induced senescence in endothelial cells through upregulation of NRF2-mediated antioxidant responses [START_REF] Sakai | Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells[END_REF].

Taken together, the present findings indicate that the old mesenteric arteries are characterized by endothelial dysfunction with blunted NO-mediated relaxation, no more EDH-mediated relaxation, and increased COX-derived EDCF responses. Furthermore, the endothelial dysfunction is associated with increased vascular ROS formation, activation of the local angiotensin system, and premature senescence in the arterial wall. Intake of the omega-3 PUFA EPA:DHA 6:1 formulation for 14 days was able to significantly improve the endothelial function by restoring NO-mediated relaxation and decreasing EDCF-mediated contractile responses. The beneficial effect of EPA:DHA 6:1 on the endothelial function is associated with the normalization of the expression level of the local angiotensin system and, subsequently, of the vascular formation of ROS and premature senescence. Rings with endothelium were prepared from the main mesenteric artery and suspended in organ baths for the determination of changes in isometric tension. (A-D) To study the endothelium-dependent relaxation, the rings were contracted with 1 µM phenylephrine (PE) before the addition of increasing concentrations of acetylcholine (ACh). (E-H) To assess the contractile responses, concentration-contraction curves were constructed in response to PE. In some baths, the rings were exposed to a pharmacological agent for 30 min before contraction.

To study the role of cyclooxygenase-derived prostanoids, the rings were incubated with indomethacin (10 µM, a nonselective COX inhibitor). To study the NO-mediated component, the rings were incubated with N ω -nitro-L-arginine (L-NA, 300 µM, an eNOS inhibitor). The EDH-mediated component was studied in the rings incubated with TRAM-34 plus UCL-1684

  This study was performed in accordance with the guidelines on animal care published by the US Institute of Health (Bethesda, MD, USA; NIH publication number 85-23, revised 1996) and the French Legislation. The protocol for this study was approved by the local Ethics Committee (Comité Régional d'Ethique en Matière d'Expérimentation Animale de Strasbourg) and authorized by the French Ministry of Higher Education, Research, and Innovation (authorization no. #7626-201611171554293).

  France), COX-2 (1/200, Cat. Ab15191, Abcam), eNOS (1/100, Cat. 610297, BD Transduction Laboratories, Le Pont de Claix, France), p16 (1/200, Cat. db018, Delta Biolabs, California, USA), p21 (1/100, Cat. Sc817, Santa Cruz Biotechnology, CliniSciences, Nanterre, France), p53 (1/100, Cat. Sc6243, Santa Cruz Biotechnology), p22 phox (1/100, Cat. Sc11712, Santa Cruz Biotechnology), p47 phox (1/100, Cat. Sc7660, Santa Cruz Biotechnology), AT1R (1/200, Cat. Sc1173, Santa Cruz Biotechnology), and ACE (1/200, Cat.250450, Abbiotec, San Diego, USA). The next day, all sections were washed with PBS before incubation with a solution of blocking buffer containing a fluorescent secondary antibody (Alexa Fluor 633 antirabbit or antimouse, Invitrogen, Fisher Scientific France, Illkirch, France) for 2 h at room temperature in the dark, followed by washing with PBS and air drying for 15-20 min. The slides were then cover-slipped with Dako fluorescence mounting solution (Ref. S3023, Dako, California,

Figure 1 :

 1 Figure 1: EPA:DHA 6:1 improves the age-related endothelial dysfunction in the main

Figure 2 :

 2 Figure 2: EPA:DHA 6:1 improves the age-related decreased NO component of the

( 1

 1 µM each, inhibitors of IKCa and SKCa, respectively). Results are expressed in % relaxations (A-D) or in grams of contraction (E-H) as means ± SEM of 10 rats per group. * P<0.05 vs. control without inhibitors.

Figure 3 :

 3 Figure 3: EPA:DHA 6:1 improves the age-related upregulation of eNOS and COX-2 and

Figure 4 :

 4 Figure 4: EPA:DHA 6:1 improves the age-related increased vascular formation of ROS

Figure 5 :

 5 Figure 5: EPA:DHA 6:1 improves the age-related upregulation of the NADPH oxidase

Figure 6 :

 6 Figure 6: EPA:DHA 6:1 improves the age-related activation of the local angiotensin

Figure 7 :

 7 Figure 7: EPA:DHA 6:1 improves the age-related upregulation of senescence markers in

Figure 1 C

 1 Figure 1

Acknowledgment:

M.A.F. was supported by a PhD fellowship from the Higher Education Commission of Pakistan.

Conflict of interest:

The authors report no conflict of interest.

Declarations of interest:

None.


Pauline Chalons

Flavie Courtaut

Emeric Limagne

Fanny F Chalmin

Emma Cantos-Villar

Tristan Richard

Cyril Auger

Philippe Chabert

Valérie Schini- Kerth

François Ghiringhelli

Virginie Aires

Dominique Delmas
email: dominique.delmas@u-bourgogne.fr

Red wine extract disrupts Th17 lymphocyte differentiation in a colorectal cancer context

Keywords: polyphenols, red wine extract, colon cancer, chemoprevention, lymphocyte Red wine extract, Polyphenols, lymphocytes Th17, Interleukins

Introduction

For several years, numerous epidemiological studies have shown that there is a strong link between inflammation and the immune system. Indeed, immune cells play a role in firstline defense following various traumas caused by infectious agents, chemical substances, physical agents or even post-traumatic tissue lesions. In contrast, inflammation has been described as an initiator event of major diseases with a significant impact in terms of public health such as cardiovascular diseases, autoimmune diseases, eye diseases, age-related diseases, and more particularly neurodegenerative diseases and the occurrence and development of cancers. In this last context, tumoral cells can produce various chemokines through different process. Among the most well-known processes, the release of interleukin-1 beta (IL-1β) following the activation of a multiprotein complex, the inflammasome, is responsible for inflammation that can be acute but also chronic or low-noise, causing many disorders including the occurrence of cancers. A second well-known process is the secretion of pro-inflammatory interleukins by immune cells that are able to overproduce various factors promoting proliferation, inflammation, neoangiogenesis. Among these proteins, we found IFN-γ, TNFα, IL-6, IL-17A, and IL-25 that strongly contribute to tumoral growth. Therefore, one of the major current issues would be to find compounds capable of modulating these production processes of pro-inflammatory substances and thus help reduce the occurrence of pathologies or reduce their progression. In this way, various studies have pointed the interest of polyphenolic compounds to prevent various diseases without toxicities.

For example, numerous studies reveal that phytoconstituents or micronutrients can protect against cancers [START_REF] Renaud | Alcohol and mortality in middleaged men from eastern France[END_REF]. Especially, Levi et al. show an inverse relation between resveratrol (a non-flavonoid polyphenol) and breast cancer risk [START_REF] Levi | Resveratrol and breast cancer risk[END_REF]. Another cohort study in Finland [START_REF] Knekt | Dietary flavonoids and the risk of lung cancer and other malignant neoplasms[END_REF] shows a link between flavonol consumption and reduced risk of lung cancer. These findings lead to the suggestion that polyphenols and flavonoids have beneficial health effects. Very recently, we have highlighted the potential anti-inflammatory effect of an extract of red wine enriched in polyphenol compounds (RWE) on macrophages [START_REF] Chalons | Study of Potential Anti-Inflammatory Effects of Red Wine Extract and Resveratrol through a Modulation of Interleukin-1-Beta in Macrophages[END_REF]. This extract was able to strongly decrease IL-1β secretion through a modulation of the expression of key proteins involved in the inflammasome complex, i.e., NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC). Moreover, we have demonstrated that RWE disrupted the priming signal and the activating signal leading to inflammasome activation in macrophages [START_REF] Chalons | Study of Potential Anti-Inflammatory Effects of Red Wine Extract and Resveratrol through a Modulation of Interleukin-1-Beta in Macrophages[END_REF]. This important way could then prevent inflammation in various contexts, but at present there is no data on the effect of a complex mixture such as a polyphenol-rich red wine extract on the immune response and its involvement in tumor progression.

Considering the fundamental role playing by the lymphocytes T in cell-mediated immunity, and more particularly by the lymphocytes T helper (Th cells) are also known as CD4 + T cells, we investigated the potential effect of a red wine extract (RWE) on Th17 immune cells that have inflammatory properties derived from their ability to produce various interleukins (i.e. IL-17A, IL-17F, IL-6, IL-21, IL-22 and TNFα). We demonstrated that RWEenriched in polyphenolic compounds reduced colorectal tumoral growth in mices which was associated with a decrease of the percentage tumor-infiltrating lymphocytes. These effects involve an alteration of the lymphocyte T differentiation into pro-inflammatory Th17 cells where the key regulators of this process, the proteins RORt (Retinoid-related Orphan Receptor gamma t) and STAT3 (Signal Transducer and Activator of Transcription 3), where affected by RWE treatment. Subsequently, this disruption of Th17 polarization leads a strongly decrease of pro-inflammatory IL-17 secretion and underlines the potential antiinflammatory properties of complex mixture of polyphenol compounds. Human colorectal cancer cell lines SW620, HCT116 and murine colorectal cancers CT26 and MC38 were obtained from the American Type Culture Collection. Cells were maintained in a 5% CO 2 humidified atmosphere at 37°C and cultured in DMEM supplemented with 10% (v/v) fetal calf serum (Dutscher). Cells were routinely tested for mycoplasma contamination using the Mycoalert Mycoplasma Detection Kit (Lonza).

Materials and Methods

Cell lines

Preparation of red wine extract

Red wine extract (RWE) was obtained from French red wine, Santenay 1er cru Les Gravières 2012 (Côte d'Or, France) selected by the BIVB (Bureau Interprofessionnel des Vins de Bourgogne) and provided by CTIVV (Centre Technique Interprofessionnel de la Vigne et du Vin). Red wine extract dry powder was prepared and analyzed as previously described [START_REF] Walter | Intake of grape-derived polyphenols reduces C26 tumor growth by inhibiting angiogenesis and inducing apoptosis[END_REF].

Briefly, phenolic compounds were adsorbed on a preparative column, then alcohol desorbed.

The alcoholic eluent was gently evaporated, and the concentrated residue was lyophilized and finely sprayed to obtain the phenolic extract dry powder. Briefly, the alcoholic eluent and water were gently evaporated using a rotary evaporator set, and the concentrated residue was deposited on the column (Diainon ® HP-20). The reservoir was filled with distilled water and the flow was adjusted to about 20 drops/min. The polyphenol fraction was eluted with an ethanol-0.1% glacial acetic acid solution (flow adjusted to 40 drops/min). The individual eluent fractions were collected and concentrated to dryness using a rotary evaporator set. One liter of red wine produced 104 g of phenolic extract, which contained 5.04 mg/g of total phenolic compounds expressed as gallic acid equivalent.

HPLC analysis

The phenolic composition of the wine samples was determined by HPLC analysis according to the method Guererro et al. [START_REF] Guerrero | Wine, resveratrol and health: a review[END_REF]. Briefly, HPLC analysis was performed on a HPLC system equipped with a diode array detector, a fluorescence detector and a C18 column (5 μm, 250 mm × 4.6 mm) for compound separation. For anthocyanins and flavonols, the mobile phase

F o r P e e r R e v i e w
consisted of water containing 5% formic acid (v/v) and methanol in various proportions at a flow rate of 1 mL/min. Anthocyanins were quantified at 520 nm as malvidin-3-glucoside and flavonols at 360 nm as quercetin-3-rutinoside. Flavan-3-ols and phenolic acids were analyzed using a gradient containing water with 2% acetic acid (v/v) and methanol-water-acetic acid (90:8:2, v/v/v). Phenolic acids were quantified at 320 nm as caffeic acid, flavan-3-ols as catechin using the fluorescence signal (excitation wavelength, 290 nm; emission wavelength, 320 nm) and stilbenes as resveratrol (excitation wavelength, 330 nm; emission wavelength, 320 nm).

Cell proliferation and viability measurements

Human and murine colorectal cancer cells were seeded in sextuplicate into 96 well-plates (1.5x10 4 cells per well) and treated for 48 and 72h at 37°C with increasing concentrations of RWE (from 1.9 to 250 μg/mL). At the end of the treatments, cells were washed with PBS, stained for 5 min with a crystal violet solution (0.5% (w/v) crystal violet in 25% (v/v) methanol) and then gently rinsed with water. Absorbances were read at 540 nm with a Biochrom Asys UVM 340 spectrophotometer, after the extraction of the dye by 0.1 M sodium citrate in 50% ethanol. The inhibitory concentrations 50 % (IC 50 ) were calculated by a 4parameter nonlinear regression with SigmaPlot version 6 software (Systat software, Inc).

Apoptosis identification

Apoptosis was determined using the annexin V-FITC and 7-aminoactinomycin D (7AAD) staining from BD Biosciences according to the manufacturer's instructions. Briefly, cells were seeded in 12-well plates (5.10 4 cells/well) 24 h before treatments. The following day, cells were treated with the indicated concentrations of RWE for 48 and 72 h. At the end of the treatments, cells were harvested, washed two times with cold PBS and then incubated for 15 min at room temperature with a mixture of annexin V-FITC/7AAD (5 µL each per sample) in 50 µL of binding buffer. At the end of the incubation, 100 µL of binding buffer was added and cells were kept on ice until analysis. Annexin V binding and 7-AAD incorporation were detected by an LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo software (Tristar, Ashland, OR, USA).

Mouse strains

Six-week-old BALB/c and C57BL/6Rj female mice were purchased from Janvier Labs (Saint-Berthevin, France). All mice were housed and maintained 22-24°C on a 12-h light-dark cycle in a designated pathogen-free area accredited by the Federation of Laboratory Animal Science Associations (FELASA). All experiments involving mice were performed in accordance with the institutional guidelines and were approved by the University of Burgundy's Ethics Committee on the Use of Laboratory Animals. Animals were provided ad libitum access to a chow diet.

Tumor growth experiments

Eight-week-old BALB/c mice were randomly divided into two experimental groups (14 mice per group). Mice were individually maintained on normal chow (control mice group) or fed per os with 100 mg/kg/day of RWE (RWE mice group), for three weeks prior to the induction of tumor formation and throughout tumors growth. To induce tumor formation, 4×10 5 mycoplasma free CT26 cancer cells were injected subcutaneously into the left flank of balb/c mice. Tumor size was monitored three times a week by caliper measurements of the widest diameter and the narrowest diameter. Time of death was considered when mice reached experimental endpoints related to graft size, animal weight and general behavior in accordance with the guidelines prescribed by the Ethics Committee at the University of Burgundy.

Isolation and analysis of tumor-infiltrating lymphocytes (TILs)

Xenograft tumors from mice groups (4 per group) described in the previous section were harvested 12 days after tumor formation induction and then dissociated in RPMI with the Lysates were centrifuged and cells were stained with an antibody cocktail containing anti-CXCR3-BV421 (CXCR3-173, BD Biosciences), anti-CD4-VioBright FITC (REA604, Miltenyi Biotech), anti-CD45RA-APC-Vio770 (30F11, Miltenyi Biotech) and anti-CCR6-PE (REA277, Miltenyi Biotech). Tumor infiltrating Th17 cells were identified as CD4 + , CD45RA -, CCR6 + , CXCR3 -. After surface staining, 2 Ml of red blood cells lysis solution (BD Biosciences) was added for 10 minutes, centrifuged (400 x g, 5 minutes) and then resuspended in flow cytometry buffer (eBioscience). All events were acquired by a BD LSR-II cytometer equipped with BD FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). The gating strategy is illustrated in Figure 5A.

In vitro Th17 cells differentiation

Naïve CD4 + T cells (CD4 + CD62L hi ) were obtained from spleens and lymph nodes of C57BL/6Rj mice. CD4 + T cells were purified from spleen and lymph nodes with anti-CD4 microbeads (Miltenyi Biotec), then were further sorted as naïve CD4 + CD62L hi T cells. Isolated naïve T cells were routinely 98% pure. Isolated naïve CD4 + T cells were stimulated with plate-bound antibodies against CD3 (145-2C11, 2 μg/mL; BioXCell) and CD28 (PV-1, 2 μg/mL; BioXCell) and polarized into effector CD4 + T lymphocyte subsets with cytokines.

Mouse, IL-6 (25 ng/mL), and TGF-β (2 ng/mL) were all purchased from MiltenyiBiotec.

Anti-IL-4 (clone 11B11; 10 µg/mL) and anti-IFNγ (clone XMG1.2; 10 μg/mL) antibodies were obtained from Bio-XCell. In experiments, RWE have been added at the final concentrations of 0.2, 2, 20 and 100 µg/ml. As previously described, cells were classically harvested on day 3 (unless otherwise specified) for detection of cytokines by ELISA and for quantitative real-time PCR (qRT-PCR) analysis as previously described [START_REF] Limagne | Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation[END_REF].

Measurement of IL-17A production

F o r P e e r R e v i e

w

After a 72-hour polarization, cell culture supernatants were assayed by ELISA for mouse IL-17A (Biolegend), according to the manufacturer's protocol.

Western Blotting analysis

Purified naïve T cells were differentiated for 6 or 24 hours into Th17 cells with or without RWE, then collected and pelleted by centrifugation (5 minutes, 450g, 4°C). Cells were lysed in boiling buffer [1% SDS, 1 mM sodium orthovanadate, and 10 mM Tris (pH 7.4)] containing protease inhibitor cocktail for 20 minutes at 4°C. Cell lysates were subjected to sonication (10 seconds at 10%) and protein concentration was assessed using the Bio-Rad DC Protein Assay Kit. Proteins were then denaturated, loaded, and separated on SDS-PAGE and transferred on nitrocellulose membranes. After blocking with 5% nonfat milk in Phosphate-Buffered Saline containing 0.1% Tween 20 (PBST), membranes were incubated overnight with primary antibody diluted in PBST containing 5% BSA: rabbit monoclonal antibodies anti-STAT3 and anti-phospho-STAT3-Tyr705 (Cell Signaling Technology), rat monoclonal antibody was used: anti-RORγt (eBiosciences), or mouse monoclonal antibody anti-β-actin (Sigma-Aldrich). Afterwards, the membranes were incubated with antibodies HRPconjugated polyclonal goat anti-mouse (Jackson ImmunoResearch), polyclonal goat antirabbit (Cell Signaling Technology) and polyclonal goat anti-rat (Santa Cruz Biotechnology) at room temperature for 1 h and developed using the ECL reagents (Supersignal West Femto maximum sensitivity substrate, ThermoFisher). Digital chemiluminescence images were captured and analyzed using the ChemiDocTM XRS + imaging system (BioRad). Image processing and analyses were carried out using Image Lab 5.2.1 build 11 Bio-Rad software.

Quantitative real-time PCR

Total RNA from T cells was extracted with TriReagent (Ambion), reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen), and was analyzed by RT-qPCR with the SYBR Green method according to the manufacturer's instructions using the 7500 Fast Real-Time PCR System (Applied Biosystems). Expression was normalized to the expression of mouse or human Actb. Primers designed to assess gene expression are as follows: Actb mus musculus 5′-atggaggggaatacagccc-3′ and 5′-ttctttgcagctccttcgtt-3′; il17a mus musculus 5'tgagcttcccagatcacaga-3' and 3'-tccagaaggccctcagacta-5' and il17f mus musculus 5′ttgatgcagcctgagtgtct-3′ and 3'-aattccagaaccgctccagt-5.

Statistical analysis

Results are shown as means ± SD for triplicate assay samples (otherwise mentioned), reproduced independently at least three times. Statistical analysis of data was carried out with Prism GraphPad6.0 Prism Software. The significance of the differences between mean values was determined by a one-way ANOVA with Holm-Sidak correction. P-values <0.05 were considered significant (*p < 0.05, **p < 0.01 and ***p < 0.001). For in vivo studies, mice survivals were estimated by the Kaplan-Meier method and were compared by the log-rank test.

Results

RWE decreases colorectal cancer cells proliferation

We have previously shown that red wine extract (RWE) can decrease the number of colonic aberrant crypt foci (AFC) in the CF-1 mouse model for AOM induced preneoplastic colonic AFC [START_REF] Mazue | Differential protective effects of red wine polyphenol extracts (RWEs) on colon carcinogenesis[END_REF] suggesting the potential interest of polyphenol extract from red wine in a precancerous context. In order to better understand the potential effects of RWE, we investigated its effects on immune cells in colorectal cancer context. Indeed, it is now clearly established that a modulation of specific immune cells can influence the development or regression of cancers. To determine the potential effect of RWE on immune cells, we have collected a French red wine, Santenay 1er cru Les Gravières 2012 (Côte-d'Or, France) from which we made a dry extract enriched in polyphenols that we so-called Red Wine Extract (RWE). In order to appreciate its polyphenolic content, we performed a qualitative and quantitative analysis by HPLC of RWE. As shown in figure 1, RWE showed high levels of phenolic acids (caftaric acid, caffeic acid), anthocyanidins (malvidin), catechins (catechin and procyanidin dimers), stilbenes (resveratrol and its glycosylated form piceid) and flavonols (quercetin). These contents are important to consider since we have previously shown that the qualitative and quantitative composition of bioactive compounds of the wine could influence the biological effects of this one. In particular, we showed that there was a synergistic effect against the proliferation of colon cancer cells resulting from an association between resveratrol and quercetin [START_REF] Mazue | Differential protective effects of red wine polyphenol extracts (RWEs) on colon carcinogenesis[END_REF]. This effect is no longer additive for a combination of resveratrol / catechin or resveratrol / catechin / quercetin. Histogram of figure 1 reveals that a high level of total resveratrol (1.92 mg/g of RWE) and quercetin (0.96 mg/g of RWE) as compared to our previous studies [START_REF] Mazue | Differential protective effects of red wine polyphenol extracts (RWEs) on colon carcinogenesis[END_REF], suggesting a potential benefit effect to induce the death of colon cancer cells. In this way, we first evaluated the RWE toxicity in our models of colon cancer cell lines (Fig. 2). Human colorectal metastatic cancer cells SW620, colorectal carcinoma cells HCT116 and murine colorectal cancer cells MC38 and CT26 were exposed to various concentrations of RWE (0.1 to 250 μg/mL) during 48 and 72 h (Fig. 2A,B). As revealed by the cytotoxic curves, RWE presented a cytotoxic activity in all colorectal cancer cell lines tested in time and concentration-dependent manner. Indeed, the inhibitory concentration of 50 % of tumor cells (IC 50 ) is more lower in human cancer cell lines after 48h of treatment than murine colorectal cancer cells (Table I), but this difference fades to 48 hours of treatment.

Secondly to determine whether these antiproliferative effects of RWE are associated with an induction of cell death, we analyzed the RWE impact on apoptosis process. To highlight this, cancer cell lines were double staining with Annexin V / 7 AAD after treatment with 100 and 200 µg/mL of RWE. Consistent with cytotoxicity curves, results show that polyphenol extract induced at early time of 48h treatment a more apoptosis in human colorectal cancer cells SW620 and HCT116 as compared to murine colorectal cancer cells MC38 and CT26 for which there are no significant differences from the control (Fig. 3A). However, at 72h we observe a strong induction of death in all colon cancer cell lines with a sharp increase in the rate of late apoptosis (Fig. 3B).

RWE decreases colorectal tumor progression in xenogreffed mice model

In a second step, we sought to correlate the inhibitory effects of proliferation and induction of apoptosis in vitro with the ability of RWE to prevent tumor progression. In order to demonstrate this, C57 BL6 mices received a fixed amount of RWE (100 mg/kg/days) for 3 weeks before receiving subcutaneous murine tumoral colon CT26 cells. Our results showed that RWE delays the growth of tumors as early as five days after tumor implantation as compared to the tumoral growth of mice controls (Fig. 4A). This difference is sharply accentuated 15 days after the injection of CT26 cells (Fig. 4A). This regression of tumor growth induced by RWE is associated with a significantly increase of survival rate in mice receiving RWE complementation (Fig. 4B).

RWE disturbs tumor-infiltrating lymphocytes

To precise the relationships between antitumoral effect of RWE and the immune cells, we evaluated the percentage of tumor-infiltrating lymphocytes. Among T lymphocytes coexist many T cell populations that are not homogenous where each subgroups present specific function and structure. We have recently shown that resveratrol was able to affect lymphocyte Th17 differentiation and subsequently disrupts interleukin-17 (IL-17) production [START_REF] Limagne | Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation[END_REF]. These resveratrol effects were associated with a reduction in various in vivo models of tumor growth and metastasis [START_REF] Limagne | Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation[END_REF]. Due to this importance of Th17 in the biological response of some polyphenols, we have investigated whether RWE could affect more specifically the CD4 + Th F o r P e e r R e v i e w cells. To further analyze these immune cells, we use polychromatic flow cytometry to sort CD4 + T cells subsets with differential expression of CD45, CXCR3 and CCR6. We observed that after 3 weeks of RWE treatment, a decreases of the percentage of tumor-infiltrating lymphocytes (CD45 + CD4 + CXCR3 -CCR6 + ) in tumor at 12 days after inoculation of tumor (Fig. 5). This significant reduction of RWE-induced TIL reduction is associated with a decrease of IL-17 as measured by ELISA method in plasma level of mice treated by RWE (not shown).

RWE counteracts IL-17 production and genes expression

Among CD4 activated cells, specific markers CXCR3-CCR6+ characterized Th17 that specific immune cells critical for progression of cancer through the production of various proinflammatory cytokines we identified the Th17 such as IL-6, IL1, IL-8 and IL-17. This last interleukin is responsible firstly for chronic and acute inflammation contributing to tumoral growth but secondly this interleukin promotes tumor angiogenesis through the stimulation of vascular endothelial growth factor (VEGF) by both tumoral cells and fibroblasts. To corraled precise the RWE effect on the specific imune T cells Th17 and its main itnerluekin 17 secretion, we have isolated naive T cells from lymph nodes and mice spleen, then they were diffrentiated invitro into pro-inflammatory Th17 with a cocktail of specific csytokines with or without various concentration of RWE during 3 days. Using ELISA method, we measured IL-17 secretion which is strongly decreases with RWE treatment in a dose dependent manner as compared to the control (Fig. 6A). This inhibition of IL-17 secretion is associated with a decrease of genes encoding this interleukin, RWE being also able to decrease mRNA levels of il-17A and iL-17F genes (Fig. 6B). As previously, immunoblotting of Th17 from in vitro differentiation of naive T cells reveals that a RWE treatment during three days decreases phosphorylation of STAT3 and subsequently strongly decreases RORt protein expression in concentration-dependent manner (Fig. 7A &B). In order to evaluated whether RWE could affect the lymphocyte T cells differentiation in animal models, we have pretreated mice with (100 mg/kg/days) for 3 weeks, and we have isolated as previously naive T cells from lymph nodes and mouse spleen before their in vitro differentiation into Th17. Interestingly, immunoblotting highlight that RWE treatment during 3 weeks significantly decreases RORt protein expression (p<0.05) and therefore the ability of naïve T cells to differentiate into pro-inflammatory Th17 (Fig. 7C & D).

Alteration of lymphocytes T differentiation by RWE in vitro and in vivo

Discussion

In the present report, we studied the effects of a red wine extract (RWE) rich in polyphenolic compounds on the immune lymphocyte T cells in vitro and in vivo and the link with the antitumoral properties of polyphenol extract. We found that antitumoral effects of RWE in vivo are associated with significant reduction of the percentage of tumor-infiltrating lymphocytes (TILs). These lymphocytes play often a dual role in the balance between proand anti tumoral effects. Thus, we highlight that RWE affects lymphocytes T into specific lymphocytes T helper called Th17 which are able to overproduce a pro-inflammatory interleukin, IL-17. We subsequently showed that RWE reduced IL-17 secretion-associated pro-inflammatory Th17. The molecular mechanism seems to involve a disruption of a specific signaling cascade where the nuclear transcriptional factor, protein RORt, which play an essential role in Th17 differentiation is strongly decrease by RWE.

The immune system can recognize many cancers (i.e. breast, ovarian, colon cancers).

Natural antitumor immunosurveillance fights against the appearance and development of cancers. Thus, it has been shown that local immunological factors may therefore have a positive effect on tumor progression or on the contrary play a deleterious role on tumor progression. Indeed, we have been able to show that a lymphocyte population called regulatory T lymphocytes could inhibit the antitumor immune response in humans as well as in animals [START_REF] Ghiringhelli | CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative[END_REF]. These regulatory T cells are able to inhibit the T dependent immune response and the increase in their number is a factor of poor prognosis in patients with ovarian cancer [START_REF] Curiel | Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival[END_REF]. Another team has shown that probably only patients with a high infiltration of CD8 effector cells into ovarian tumor and few regulatory T cells would have a good prognosis [START_REF] Sato | Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer[END_REF].

Conversely, a deleterious role of inflammation and the immune system is seen added to all these mechanisms that can pervert the immune system including via mechanisms involving the production of pro-inflammatory cytokines such as TNF, IL-6 and IL-1β. These cytokines induce a modification of the T cell polarization towards a Th17 polarization (IL-17 producing cells). This population exerts a protumoral role by increasing tumor angiogenesis and making tumor cells resistant to cell death. Therefore, by decreasing the production of proinflammatory substances such as IL 17, RWE is able to reduce tumor growth but also angiogenesis. These results allow us to provide a possible explanation for the observation of many teams including Schini-Kerth's team who showed in a model of colon cancer that red wine extract reduced neoangiogenesis [START_REF] Walter | Intake of grape-derived polyphenols reduces C26 tumor growth by inhibiting angiogenesis and inducing apoptosis[END_REF]. In fact, the modulation of immune cells could appear as the upstream element of the antitumor effects observed for the RWE but also for many polyphenols. Indeed, we have recently shown that resveratrol was able to modulate lymphocytes T differentiation into Th17 in similar manner as metformin [START_REF] Limagne | Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation[END_REF]. The antitumoral effect of resveratrol was dependent of Th17-IL-17 system. Indeed, using IL-17 knock down mices where the antitumoral resveratrol activity observed in normal mice was abolished in IL-17 KO mice [START_REF] Limagne | Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation[END_REF]. Furthermore, resveratrol effect on the IL-17 production contributes to reduce VEGF levels and subsequently contributes to strongly decrease the number of pulmonary metastasis in tumorigenesis-induced mice [START_REF] Limagne | Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation[END_REF].

The molecular mechanism of antitumoral effect and more specifically the RWE-induced immunomodulation seems more complicated, because of the relatively complex composition of the mixture (Fig. 1) that contains a high number of polyphenolic compounds that may interact with each other or target various signaling pathway actors. We previously demonstrated that the qualitative and quantitative composition of a wine in bioactive molecules such as polyphenolic compounds is crucial in the biological effects that can be observed and that they are antagonistic or synergistic [START_REF] Mazue | Differential protective effects of red wine polyphenol extracts (RWEs) on colon carcinogenesis[END_REF]. We previously showed that resveratrol and quercetin combined exhibited a synergistic effect inducing apoptosis, antiproliferative effects, and cell cycle disruption compared to the compounds alone in colon cancer cells. Interestingly, in this same study, the combination of resveratrol+catechin or resveratrol+catechin+quercetin produced only an additive effect on the inhibition of tumor cell proliferation or on cell cycle arrest, and catechin+quercetin did not induce a synergistic effect on apoptosis [START_REF] Mazue | Differential protective effects of red wine polyphenol extracts (RWEs) on colon carcinogenesis[END_REF]. In this way some reports have shown a potential effect of quercetin on Th17 in some models of inflammation. For example, quercetin was able to reduces Th17 cell polarization through an inhibition of a mitogen-activated protein kinase Toll-like receptor 4 (MAPK-TLR4) signaling pathway and subsequently decrease IL-17 production in a model of dental pulpitis [START_REF] Hashemi | Quercetin Decreases Th17 Production by Down-Regulation of MAPK-TLR4 Signaling Pathway on T Cells in Dental Pulpitis[END_REF]. Interestingly, in a model of rheumatoid arthritis using osteoclasts, quercetin reduces Th17differentiation and IL-17-stimulated RANKL (Receptor Activator of Nuclear factor kappa-B Ligand) production in RA-FLS (Rheumatoid Arthritis-Fibroblasts- Like Synoviocytes) and IL-17-stimulated osteoclast formation [START_REF] Kim | Quercetin, a Plant Polyphenol, Has Potential for the Prevention of Bone Destruction in Rheumatoid Arthritis[END_REF]. In similar manner, catechin, and more particularly epigallocatechin-3-gallate (EGCG) suppressed expansion and cell cycle progression of naïve CD4 + T by modulating cell cycle-related proteins. EGCG also inhibited naive CD4 + T-cell differentiation into Th1 and Th17 effector subsets by impacting their respective signaling transducers and transcription factors in some models of autoimmune diseases [START_REF] Yang | EGCG attenuates autoimmune arthritis by inhibition of STAT3 and HIF-1alpha with Th17/Treg control[END_REF][START_REF] Lee | Epigallocatechin-3-gallate ameliorates autoimmune arthritis by reciprocal regulation of T helper-17 regulatory T cells and inhibition of osteoclastogenesis by inhibiting STAT3 signaling[END_REF][START_REF] Wu | Green tea EGCG, T-cell function, and T-cell-mediated autoimmune encephalomyelitis[END_REF][START_REF] Byun | Epigallocatechin-3-gallate ameliorates both obesity and autoinflammatory arthritis aggravated by obesity by altering the balance among CD4+ T-cell subsets[END_REF].

But in the concept of immunosurveillance in a cancerous context, various other immune cells play key roles such as cytotoxic T cells (CTL or CD8 + T cells) that are able to induce the death of infected somatic cells or tumor cells. The activation and differentiation of CD8 + cells is an essential step for the establishment of a response against intracellular pathogens and against tumor cells. For example, in a tumor context, the polyphenol resveratrol led to an increase in the number of cytotoxic T-lymphocyte associated protein 4 (CTLA-4; also known as CD152)-positive cells and in the gene expression levels of CTLA-4, FoxP3, IL-10 and TGF-β these two last TGF-β and IL-10 being anti-inflammatory cytokines and controlling pathogenic, as well as aging-associated inflammatory conditions [START_REF] Weng | The phytogestrogenic stilbenes, arachidin-1 and resveratrol, modulate regulatory T cell functions responsible for successful aging in aged ICR mice[END_REF]. In a same manner, other immune cells such as regulatory T cells (Treg cells) appear essential in cancer progression, since the presence of Treg cells is likely to correlate with cancer progression [START_REF] Perez | CD4+CD25+ regulatory T-cell frequency in HER-2/neu (HER)-positive and HER-negative advanced-stage breast cancer patients[END_REF], and specific depletion of Treg cells markedly inhibits tumor growth and maintains a strong and persistent antitumor immune response [START_REF] Knutson | IL-2 immunotoxin therapy modulates tumor-associated regulatory T cells and leads to lasting immune-mediated rejection of breast cancers in neu-transgenic mice[END_REF]. Again many polyphenols can act on these immune cells [START_REF] Yu | Epigallocatechin-3-gallate Prevents Triptolide-Induced Hepatic Injury by Restoring the Th17/Treg Balance in Mice[END_REF][START_REF] Yang | Quercetin attenuates collagen-induced arthritis by restoration of Th17/Treg balance and activation of Heme Oxygenase 1-mediated anti-inflammatory effect[END_REF]. Additional studies are now in progress to determine the impact of a polyphenol-enriched extract on the different subsets of immune cells and more particularly on T cells that play a central role in cell-mediated immunity. Red Wine extract was able to alter differentiation of lymphocyte T cells into proinflammatory Th17 through an alteration of nuclear transcriptional factor, the protein RORt. Subsequently, Red Wine Extract disrupts pro-inflammatory interleukin IL-17. These events are associated in vivo with a decrease of tumor-infiltrating lymphocytes and with a reduction of colorectal tumor growth.

  TM dissociator (Myltenyi Biotec) according to the manufacturer's protocol.

  Differentiation of lymphocytes T is mainly under the control of signaling pathway involving key proteins such as the homodimer STAT3 and the nuclear transcriptional factor RORt. In fact, the phosphorylation of STAT3 stabilizes the protein. Once phosphorylated, STAT3 is translocated into the nucleus to active the transcription of gene encoding the protein RORt which in turn will activate the transcription of genes encoding interleukins, particularly IL-17.

  Author contributions : P.C. performed the experiments and analyzed the data; F.C. performed immunological detection; E.L., F.C. performed Th17 isolation; E.C.V. and T.R.

  HPLC analysis; C.A, P.C. and V.S.K. performed RWE production and analyzed the data; V.A. and F.G. helped in setting up the project and analyzed the data; V.A. and D.D. revised the manuscript; D.D. supervised the overall project.

Figure 1 .

 1 Figure 1. HPLC quantification of red wine extract (RWE) obtained from the Santeney

Figure 2 .

 2 Figure 2. Concentration-dependent inhibition of colon cancer cells proliferation by

Figure 3 .

 3 Figure 3. RWE-induced apoptosis in colon cancer cell lines. After 24h of culture, SW620,

Figure 4 .

 4 Figure 4. RWE reduced colorectal tumor growth and increased survival level of mice.

Figure 5 .

 5 Figure 5. RWE decreases Tumor-Infiltrating Lymphocytes (TILs). Eight-week-old

Figure 6 .

 6 Figure 6. RWE-induced interleukin 17 inhibition in lymphocytes Th17. CD4 + T cells

Figure 7 .

 7 Figure 7. RWE affects key regulator protein of Th17 differentiation. A) CD4 + T cells

  Figure 2

Table I . Determination of IC 50 for RWE on the four colorectal cell lines tested. 72h 48h IC 50 of RWE (µg/ml)

 I

	Figure 1
	Cell lines Cell lines	72h IC 50 of RWE (µg/ml) 48h	18,75
	SW620 HCT116 MC38 SW620 HCT116 MC38 mg / g RWE 5,04	123.7 ± 11.9 200.0 ± 4.3 213.5 ± 30.4 123.7 ± 11.9 200.0 ± 4.3 213.5 ± 30.4 12,74 4,8 2,4	60 ± 15.7 74.5 ± 11 .6 200 ± 6.6 60 ± 15.7 74.5 ± 11 .6 200 ± 6.6 0,24 1,68 3,6 2,4	3,36
	CT26 CT26	121.0 ± 9.8 121.0 ± 9.8 F o r 150 ±30.0 150 ±30.0
		P
			e e r
			R e v
			i e w

Acknowledgements:

The authors are supported by grants from Bourgogne Franche-Comté Regional Councils (CRBFC), the FEDER and the "Bureau Interprofessionnel des Vins de Bourgogne" (BIVB).

Conflicts of Interest: the authors declare no conflict of interest.


Camélia Kitoun

Matthieu Fonvielle

Nicolas Sakkas

Manon Lefresne

Fabiola Djago

Quentin Blancart Remaury

Pauline Poinot

Michel Arthur

Mélanie Etheve-Quelquejeu

Laura Iannazzo

Phosphine-Mediated Bioconjugation of the 3'-End of RNA

come

Phosphine-Mediated Bioconjugation of the 3'-end of RNA Camélia Kitoun, † Matthieu Fonvielle, ‡ Nicolas Sakkas, † Manon Lefresne, † Fabiola Djago, § Quentin Blancart Remaury, § Pauline Poinot, § Michel Arthur, ‡ Mélanie Etheve-Quelquejeu,* † Laura Iannazzo* † † Université de Paris, UMR CNRS 8601, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, F-75006 Paris, France. ‡ INSERM, Sorbonne Université, Université de Paris, Centre de Recherche des Cordeliers (CRC), F-75006, Paris, France. § Institut de Chimie des Milieux et Matériaux de Poitiers IC2MP, Université de Poitiers, UMR 7285, 86073, Poitiers, France.

ABSTRACT: Staudinger ligation is an attractive bioorthogonal reaction which has been widely used to tag proteins, carbohydrates and nucleic acids. Here, we explore the traceless variant of the Staudinger ligation for 3'-end modification of oligoribonucleotides. An azido-containing dinucleotide was used to study the ligation. Nine phosphines containing reactive groups, affinity purification tags or photo-switch probes have been successfully obtained. The corresponding modified dinucleotides were synthesized and characterized by LC/MS. Mechanistic interpretations of the reaction are proposed, in particular, the unprecedented formation of an oxazaphospholane nucleotide derivative, which was favored by the vicinal position of 2'-N3 and 3'-OH functional groups on the terminal ribose has been observed. The post-functionalization of a 24-nt RNA with a photoactivable tag is also reported.

Bioconjugation reactions 1 developed in the last decade enabled the chemical labeling of biomolecules to enrich their functions and properties. Owing the complexity of the biomolecules, due to the presence of reactive groups and their sensitivity to non-physiological pH or temperature, the bioconjugation reactions have to be chemoand regio-selective, and compatible with nearly physiological conditions in aqueous media. One strategy to enhance the selectivity of a bioconjugation reaction is to first install a unique functional group onto the biomolecule, and then use site-selective type reactions to covalently link the desired tags. A number of site-selective reactions targeting non-native functional groups of biomolecules have been reported so far: i) the condensation between a carbonyl and hydrazines, hydrazides or alkoxyamines; 2 ii) metal-mediated reactions, such as Alkyne-Azide Cycloadditions (CuAAC or NiAAC), 3 Sydnone-Alkyne Cycloaddition (CuSAC), 4 or Palladium-Catalyzed Cross-Coupling Reactions; 5 iii) strained-promoted cycloadditions, such as dipolar cycloadditions with constrainted alkynes (SPAAC), 6 or Inverse Electron Demand Diels-Alder reactions (IEDDA); 7 iv) photoclick reactions, 8 and finally v) chemical ligation such as Native Chemical Ligation (NCL) 9 or Staudinger ligation. 10 The latter reaction is an attractive bioorthogonal conjugation reaction, especially for in vitro and in vivo applications, due to the absence of a metal catalyst and to its capacity to form native amide bond between the tag and the biomolecule. The traceless variant of the Staudinger ligation, independently developed by Raines 11 and Bertozzi, 12 is even more interesting, since it leads to the elimination of the phosphine oxide from the final product. These new phosphines developed by Bertozzi and coworkers, 12 contain an acyl component, which is attached through a cleavable linkage (an ester bond) at the ortho-position of the aryl group of the phenylphosphine. These reagents have been designed to avoid the oxidation of the phosphine under ambient conditions, to introduce conformational rigidity and to promote a six-membered transition state during the acyl transfer. To access a transition state with a smaller ring, Raines and co-workers replaced the O-phenyl group by a single methylene moiety and increased the yield of the traceless ligation to couple peptides and azides. 13 This methodology has been employed for the bioconjugation of polysaccharides, 14 peptides, 15 or proteins. 16 Application of the Staudinger-type ligation to nucleoside or nucleotide modification has been mostly restricted to the synthesis of puromycin analogues, 17 the functionalization with fluorescent probes 18 and the introduction of affinity purification tags. 19 The application of the traceless version of the Staudinger ligation has been even more restricted, and, to the best of our knowledge, only reported for the functionalization of a protected 5'-azido-2'-deoxyadenosine. 12 Since chemistry driven studies of the biological role of RNAs requires new synthetic methodologies to modify their structures, and because no example depicts the modification of RNA by traceless Staudinger ligation, we decided to explore this site-selective reaction as a post-synthetic methodology for the functionalization of RNAs with a variety of functional groups.

Our strategy to access to functionalized RNA relies on the enzymatic ligation of a 5'-phosphorylated dinucleotides (pdC-pA) to an oligoribonuleotide by the T4 RNA ligase. The presence of a 2'-deoxycytidine at the 5' position of the dinucleotide facilitates its synthesis and is compatible with the specificity of the T4 RNA ligase. The presence of a 2'-deoxynucleotide at the penultimate position of the ligation product was previously reported to have little impact on the biological function of the final RNA. 20 To study the bioconjugation of the 3'-terminal RNA position, we selected as a model, the dinucleotide 1 containing an azido group at the 2' position of the terminal adenosine. The second partner of the reaction was the tag-substituted phosphine. Among the modified phosphine reagents developed in the literature for the traceless Staudinger ligation, we choose phenolic ester 2a and alkyl thioester 3 since they were reported to provide highest ligation yields. 21 When dinucleotide 1 was treated in presence of 10 equivalent of methylester phenolic phosphine 2a (synthesized as shown in Scheme 2) and heated for 4 h at 70°C followed by the addition of water and 2 h of stirring at 70 °C, the full conversion of the starting dinucleotide was observed (Scheme 1, entry 1). These conditions lead to the formation of the ligated compound 4a in 22% yield in competition with the reduced derivative 6. In addition, a cyclic oxazaphospholane derivative 5a was obtained in 28% yield. Evidence for the formation of compound 5a was gathered by monitoring the reaction by 31 P NMR spectroscopy (See supporting information S70). After 55 min of reaction in DMF/D2O (9/1, v/v), a growing signal at + 40.6 ppm was observed compared to the characteristic peak of the phosphine 2a at -15.8 ppm and occurrence of oxidized species. The formation of such cyclic derivative could be explained by the 1,2-cis position 22 of the 2'-N3 function and the 3'-OH hydroxyl on the ribose. The modification of the reaction conditions with a longer reaction time or irradiation under micro-waves did not allow to increase the yield of the ligated compound 4a and dramatically increased the formation of the oxazaphospholane 5a (See supporting information S56).

Scheme 1. Reaction of dinucleotide 1 with phenolic ester 2a and alkyl thioester 3 substituted with a methyl group.

We next performed the same ligation from the dinucleotide 1 with the alkyl thioester phosphine 3 (the commercially available (diphenylphosphino)methanethiol) described by Raines. 13 Treatment with DABCO was first applied to remove the borohydride group which prevented the oxidation of the phosphine. In this latter condition, the cyclic oxazaphospholane derivative 5a was not observed in the reaction mixture. The reduced compound 6 was obtained as the major product (77% yield), whereas the expected dinucleotide 4a was formed in 23% yield (Scheme 1, entry 2).

To expand the scope of the reaction, we selected the phosphine phenolic ester derivatives due to their stability and easy functionalization. A range of substituted phosphines was synthetized by coupling the commercially available 2-hydroxyphenyl diphenylphosphine with the corresponding carboxylic acid in presence of DCC and DMAP in DCM at room temperature for 16 h. Eight phosphines were obtained in 18 to 95% yield (Scheme 2). Scheme 2. Synthesis of phosphines 2a-h. Phosphine 2j containing a sydnone substituent, recently reported as a suitable partner for click and release experiments for bioconjugation, 4,23 was synthesized according to the synthetic route described in Scheme 3. Iminosydnone 7 was prepared following standard procedure 23 and activated by a 2-step strategy 24 to afford the corresponding carbonyl-imidazolium salt 8 in 75% yield. The sydnone compound 8 was then treated in the presence of DIPEA and unprotected phosphine 2i in DCM to lead to the phosphine 2j in 54% yield. Scheme 3. Synthesis of phosphine 2j. Phosphines 2a-j containing representative functionalities were submitted to ligation conditions (Scheme 4). When phosphine-containing phenyl or pyridine rings (2b, 2c) were used for bioconjugation reaction with dinucleotide 1, compounds 4b and 4c were obtained in 77% and 24% yield respectively. Photoactivable dinucleotides 4d and 4e have been prepared in 12% and 76% yield, respectively, using phosphine 2d 25 and 2e. A biotin moiety, used for analysis and purification of biomolecules, was introduced in the 2'-position of the terminal ribose, in 59% yield. A photoswitch tag has also been ligated and diazobenzene phosphine 2g led to the formation of the corresponding modified dinucleotide 4g in 47% yield. The ligation was then applied to access to the dinucleotide-sydnone conjugate 4j in 86% yield. These results show that the functionalization in 2'-position of the terminal ribose of an unprotected dinucleotide is compatible with ester phenolic phosphine containing a variety of tags. [START_REF]The use of a 2'-deoxynucleoside is recommended on the penultimate position to avoid the break of the phosphodiester bridge, probably triggered by the formation of the oxazaphospholane[END_REF] To go further, we applied this reaction to post-functionalize a 24-nt RNA containing an azido group at 2'-position of the terminal ribose. The 2'-azido-RNA 10 was obtained by enzymatic ligation of dinucleotide 9 (pdCpA-2'-N3 ) 27 to a 22-nt RNA using the T4 RNA ligase RNA molecule (See supporting information S14). The sequence of the RNA has been chosen to mimic the acceptor arm of Ala-tRNA Ala . The oligonucleotide 10 was then treated by an excess of phosphine 2e (100 equiv.) containing a benzophenone tag. After 4 h stirring at 70 °C in a 9/1 mixture of DMF and water, followed by the addition of water and 2 h stirring at 70 °C, the reaction lead to the formation of the functionalized RNA 11 in 7% yield (Scheme 5). This was confirmed by mass spectrometry (MALDI analysis, See supporting information S66-69). Scheme 5. Bioconjugation of 24nt-RNA.

A putative mechanism accounting for our experimental results is proposed in Scheme 6. After formation of the iminophosphorane intermediate I, Staudinger reduction could lead directly to the formation of 2'-NH2 dinucleotide II. This reaction occurred in competition with the acyl transfer from the phosphine to the nitrogen of the iminophosphorane (pathway A). This step is promoted by the sixmembered ring formed during the transition state. Intermediate III could then be hydrolyzed affording the expected ligation product IV. Moreover, the nitrogen of the iminophosphorane and the hydroxyl at the 3' position were in the cis configuration, which favored the ring-closing step (pathway B) and gave access to the oxazaphospholane intermediate V. This reactivity was reported by Bernardi and co-workers, who investigated the traceless ligation of glycofuranosyl azides. 22 Triaryliminophosphorane moiety 28 could promote the deprotonation of the alcohol at the 3'-position and favor the nucleophilic attack of the phosphorus. Then, the hydrolysis of V could provide reduced compound II and the oxidized phosphine. In conclusion, we have reported the first application of the traceless Staudinger ligation to the post-functionalization of RNA. We synthesized a series of ester phenylphosphines, which contained a variety of tags, such as a Biotin, photoswitch and photoactivable moieties, and a sydnone. We studied the traceless variant of the Staudinger ligation on a model dinucleotide and showed that the reaction provided moderate to good yields, depending on the nature of the ester-phenyl phosphine. We extended this methodology for the bioconjugation of a 24nt RNA strand with a photoactivable benzophenone probe.

G G C U C C A C C U C G A U U C C G G G G . A G G C U C C A C C U C G A U U C C G G G G .

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Characterization of phosphines 2a-i (PDF) Synthesis and characterization of phosphine 2j (PDF) Synthesis and characterization of dinucleotide 1 (PDF) Synthesis and characterization of dinucleotide 9 (PDF) NMR spectra (PDF) LCMS analyses (PDF) FAIR Data is available as Supporting Information for Publication and includes the primary NMR FID files for compounds 1, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 7,

a

  Scheme 4. Substrate scope.

a

  Product yield based on relative integration in HPLC at 254 nm.

  Scheme 6. Proposed mechanism.

ACKNOWLEDGMENT

This research was funded by grants from the Agence National de la Recherche (ANR), Project SyntRNA [ANR-17-CE07-0041-02].

AUTHOR INFORMATION

Corresponding Author *E-mail: laura.iannazzo@parisdescartes.fr; Phone: (33) 1 76 53 42 50; *E-mail: melanie.etheve-quelquejeu@parisdescartes.fr; Phone: (33) 1 76 53 42 22.