Aloïs Dusuel
email: alois.dusuel@protonmail.ch

Valérie Deckert
email: valerie.deckert@u-bourgogne.fr

Jean-Paul Pais De Barros

Kevin Van Dongen
email: kevin.van-dongen@u-bourgogne.fr

Hélène Choubley
email: helene.choubley@u-bourgogne.fr

Émilie Charron
email: emilie.charron@u-bourgogne.fr

Naïg Le Guern
email: naig.le-guern@u-bourgogne.fr

Jérôme Labbé
email: jerome.labbe@u-bourgogne.fr

Stéphane Mandard
email: stephane.mandard@u-bourgogne.fr

Jacques Grober
email: jacques.grober@u-bourgogne.fr

Laurent Lagrost
email: laurent.lagrost@u-bourgogne.fr

Thomas Gautier
email: thomas.gautier@u-bourgogne.fr

Human cholesteryl ester transfer protein lacks lipopolysaccharide transfer activity, but worsens inflammation and sepsis outcomes in mice

Keywords: ALT, Alanine Aminotransferase, BPI, Bactericidal / Permeability Increasing protein, CD14, Cluster of Differentiation 14, CE, Cholesteryl Ester, CETP, Cholesteryl Ester Cholesterol, Infection, Inflammation, Lipoproteins, Plasma Lipid Transfer Proteins, Endotoxins, Lipopolysaccharide (LPS), Sterol metabolism, Sepsis, Bacteremia

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INTRODUCTION:

Sepsis is a life-threatening condition characterized by a suspected or proven infection followed by a systemic inflammatory response syndrome (SIRS). The dysregulated inflammatory response of the host can lead to multiple organ failure (i.e. the main feature of severe sepsis) which may cumulate with persistent hypotension in the most advanced stage of septic shock. In 2011, sepsis was the most expensive condition treated in the U.S., with annual hospital costs exceeding $ 20 billion [START_REF] Torio | National Inpatient Hospital Costs: The Most Expensive Conditions by Payer, 2013: Statistical Brief #204. Healthcare Cost and Utilization Project (HCUP) Statistical Briefs[END_REF]. Even if related infection cannot be identified in a systematic way in the hospital, bacteremia is one major and leading trait of sepsis. Gramnegative bacteria, which are identified in 62% of cases [START_REF] Vincent | International study of the prevalence and outcomes of infection in intensive care units[END_REF], can release lipopolysaccharides (LPS) in bloodstream. LPS, or endotoxins, are amphipathic molecules with pro-inflammatory properties. They activate TLR-4/CD14 complex in leukocytes, leading to cytokine release and SIRS [START_REF] Matsuda | Systemic inflammatory response syndrome (SIRS): molecular pathophysiology and gene therapy[END_REF].

Importantly, the bulk of LPS cannot be metabolized in the body, with the exception of some deacylation reactions that may occur (4). To get rid of LPS, mammal body relies on one main pathway through which LPS can be disaggregated, transported in plasma and excreted in the bile. This pathway, known as the reverse LPS transport (RLT) pathway consists in 3 major steps: 1) LPS disaggregation and binding to circulating lipoproteins, 2) uptake of lipoprotein-bound LPS by the liver, and 3) LPS excretion in the bile [START_REF] Gautier | Plasma PLTP (phospholipid-transfer protein): an emerging role in "reverse lipopolysaccharide transport" and innate immunity[END_REF]. Importantly, whereas lipoproteins are recognized as the main carriers of LPS in plasma, RLT involves key proteins that belong to the lipid transfer / LPS binding protein (LT/LBP) gene family and play differential roles in LPS metabolism. It includes LBP, BPI, PLTP, and CETP.

Lipopolysaccharide Binding Protein (LBP) is an acute phase protein that disaggregates, binds, and presents LPS to the TLR-4/CD14 complex and contributes to monocyte activation [START_REF] Schumann | Structure and function of lipopolysaccharide binding protein[END_REF]. Bactericidal / Permeability-Increasing protein (BPI) is stored in primary granules of polymorphonuclear leukocytes. BPI inhibits bacterial growth by binding LPS and forming complexes directly to the outer membrane of bacteria, thus preventing leukocyte activation [START_REF] Elsbach | The bactericidal/permeability-increasing protein (BPI) in antibacterial host defense[END_REF][START_REF] Tobias | Lipopolysaccharide (LPS)-binding proteins BPI and LBP form different types of complexes with LPS[END_REF]. In human and animal studies, recombinant BPI was found to be relevant in binding and neutralizing LPS [START_REF] Levy | Bactericidal/permeability-increasing protein in host defense and its efficacy in the treatment of bacterial sepsis[END_REF]. Unlike LBP and BPI, Phospholipid Transfer Protein (PLTP) has been initially mainly studied in the context of lipid transport, atherosclerosis and cardiovascular diseases. Importantly however, PLTP was lately reported as a major component of RLT. Among LPS detoxifying properties, its ability to bind and transfer LPS between lipoproteins [START_REF] Hailman | Neutralization and transfer of lipopolysaccharide by phospholipid transfer protein[END_REF], to foster LPS excretion in the bile [START_REF] Gautier | Plasma PLTP (phospholipid-transfer protein): an emerging role in "reverse lipopolysaccharide transport" and innate immunity[END_REF], and to kill bacteria [START_REF] Deckert | Recombinant human plasma phospholipid transfer protein (PLTP) to prevent bacterial growth and to treat sepsis[END_REF]. Finally, Cholesteryl ester transfer protein (CETP) was mostly studied in the context of cholesterol metabolism and atherosclerosis because of its ability to exchange cholesteryl esters (CE) and triglycerides (TG) between high density lipoprotein (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL). However, and unlike LBP, BPI and PLTP which are today well recognized as LPS binding proteins, the role of CETP in LPS metabolism remains puzzling.

In transgenic mice expressing human CETP under the control of its natural flanking regions, LPS administration leads to a decrease in plasma CETP activity and liver CETP mRNA levels [START_REF] Masucci-Magoulas | Decreased cholesteryl ester transfer protein (CETP) mRNA and protein and increased high density lipoprotein following lipopolysaccharide administration in human CETP transgenic mice[END_REF]. In patients with sepsis, a pronounced reduction of CETP plasma concentration on the first day of admission was associated with a higher mortality rate [START_REF] Grion | Lipoproteins and CETP levels as risk factors for severe sepsis in hospitalized patients[END_REF]. Although these earlier data suggest that CETP might play a role in LPS transport and metabolism, it is not known whether altered CETP levels in patients is a cause or a consequence or sepsis severity. In addition, it remains unclear at this stage whether CETP can actually modulate inflammation and sepsis in vivo, and whether it might be involved in LPS transport either directly through its ability to bind LPS with a very low affinity [START_REF] Clark | Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection[END_REF], or indirectly through its ability to modify lipoprotein structure, composition and kinetics [START_REF] Ikewaki | Delayed catabolism of high density lipoprotein apolipoproteins A-I and A-II in human cholesteryl ester transfer protein deficiency[END_REF].

In the present study, we investigated whether CETP may contribute to binding and transfer of LPS in plasma, in particular in comparison with PLTP as a main LPS transfer protein in the bloodstream. To this end, we set up in vitro methods to measure LPS transfer activity between lipoproteins. In parallel, the contribution of CETP to resistance against sepsis and its harmful consequences was investigated by comparing wild-type (WT) and heterozygous human CETP-expressing (CETPTg) mice in two models of acute inflammation, i.e. after intravenous (iv) injection with purified LPS, or induction of polymicrobial infection as obtained after cecal ligation and puncture (CLP). Here, concentration of LPS in plasma, liver and bile compartments could be monitored for the first time using a direct mass assay of LPS.

MATERIAL AND METHODS:

LPS transfer activity

Preparation of donor Sepharose-CNBr-HDL-LPS-DOTAGA-Bodipy-NCS complex.

Conjugation of DOTAGA-Bodipy-NCS to LPS. DOTAGA-Bodipy-NCS was obtained as previously described [START_REF] Bernhard | First bodipy-DOTA derivatives as probes for bimodal imaging[END_REF]. LPS from Salmonella enterica serotype Minnesota (Sigma-Aldrich) was resuspended in sodium bicarbonate buffer at a concentration of 10.0 g/L. One equivalent of DOTAGA-Bodipy-NCS was suspended in DMSO at a concentration of 10.0 g/L and was incubated with LPS for one hour at 37°C and 500 rpm shaking. Labeled LPS was separated from free DOTAGA-Bodipy-NCS by liquid chromatography using a Superdex 75 column (GE Healthcare) and dialyzed against PBS.

Conjugation of CNBr Activated Sepharose to HDL. HDL were isolated from human plasma provided by the French Blood Establishment (EFS). CNBr activated sepharose 4B (GE Healthcare, 0.86 g) was suspended in 3.0 mL of 1.0 mM HCl buffer. The gel was then washed with coupling buffer (NaHCO3 0.2 M, NaCl 0.5 M, pH 8.3) and incubated in the presence of human HDL (27.0 mg protein) overnight at 4°C. During incubation, the tubes were continuously inverted to avoid sedimentation. Excess of ligand was washed with coupling buffer, and unbound sites were blocked with Tris-HCl buffer (0.1 M). The gel was then washed with acetate buffer (0.1 M) and coupling buffer 3 times consecutively, before being equilibrated with PBS.

Conjugation of CNBr Activated Sepharose to VLDL. VLDL (7.15 mg protein) were isolated from human plasma and coupled to CNBr activated sepharose 4B (0.28 g) as described above for HDL.

Binding of LPS-DOTAGA-Bodipy to Sepharose-CNBr-Lipoproteins. Sepharose-CNBr-HDL or Sepharose-CNBr-VLDL was incubated with LPS-DOTAGA-Bodipy for one hour at 37°C with continuous inversion (1.0 mg of LPS for 2.0 mg of HDL protein or 3.6 mg VLDL protein). Tubes were centrifuged (1 min, 14.000 g) and pellets were washed with PBS 3 times consecutively to discard unbound LPS-DOTAGA-Bodipy-NCS.

Incubation of Sepharose-CNBr-Lipoprotein-LPS-DOTAGA-Bodipy with plasma.

Donor Sepharose-HDL (20µg protein) or Sepharose-VLDL (16.8 µg protein) with bound LPS-DOTAGA-Bodipy were incubated in the presence of 16 µl (≈0.55 mg of total protein) of plasma from WT, CETPTg or PLTPKO mice as a source of endogenous acceptor lipoproteins and lipid transfer proteins in a final volume of 100µl PBS. The mixtures were incubated at 37°C under continuous inversion. At indicated time point, tubes were centrifuged during 1min at 14.000 g to pellet donor lipoprotein complex. Fluorescence of transferred LPS-DOTAGA-Bodipy was measured in supernatant with a Wallac Victor 3 (ThermoFisher) plate reader (excitation 485 nm, emission 535 nm).

Animals

The experimental protocol was approved by the University of Burgundy's Ethics Committee on the Use of Laboratory Animals. PLTP-deficient (PLTPKO) mice [START_REF] Jiang | Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels[END_REF] and heterozygous transgenic mice expressing human CETP (CETPTg mice) [START_REF] Jiang | Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences[END_REF] were bred on a homogenous C57BL/6J genetic background.

We used male CETPTg, PLTP KO and wild-type (WT) C57BL/6J control mice aged from 8 to 16 weeks.

Animals had unlimited access to water and standard chow diet (A03-10, Safe) and were housed under a 12h day-night cycle.

Blood and organ sampling

Blood was collected with heparin via retroorbital puncture for intermediate time points, and via cardiac puncture for final time points. Plasma was isolated by centrifugation (10 min at 6000 g). Plasma lipoproteins were isolated by sequential ultracentrifugation (Apolipoprotein B-containing lipoproteins (LpB): d<1.063; High Density Lipoproteins (HDL): 1.063<d<1.210). Liver was collected after cardiac puncture and sacrifice, and immediately placed in liquid nitrogen before storage at -80°C.

Injection of purified LPS

To assess the kinetics of LPS clearance and inflammation, a first set of WT and CETPTg mice were injected intravenously (iv) with a single dose (1.0 mg / kg body weight) of purified LPS (Escherichia coli O55:B5, Sigma Aldrich). Blood was sampled before (0min), and 30min, 1h, 3h, 6h, 24h after LPS injection on each animal. A second set of WT and CETPTg mice was injected iv with a single dose (15 mg/kg body weight) of purified LPS (Escherichia coli O55:B5, Sigma Aldrich) and monitored during 10 days to assess survival. In experiments assessing cytokine burst as well as alterations in plasma lipoprotein profile and leukocyte counts in response to acute endotoxemia, samplings were conducted at 0 and 2 hours after LPS injection.

Cecal ligation and puncture (CLP)

Mice were anesthetized with isoflurane (Vetflurane, Virbac) and placed on a heating plate during surgery. For each animal, abdomen was shaved and disinfected with ethanol and sterile gauzes before performing midline laparotomy. The cecum was 1) exteriorized, 2) ligated at 75% from its distal end using 4-0 surgical suture, 3) perforated through-and-through with a 21-gauge needle to 4) extrude a small amount of luminal content. The surgical procedure for sham animals only consisted in step 1). After gently replacing the cecum in the peritoneal cavity, abdominal muscle and skin were sutured using 6-0 surgical suture and wound clips, respectively. Mice were injected subcutaneously with 0.4 mL of saline to compensate hydric loss that occurred during the procedure, and awakened in warm conditions using an infrared bulb during one hour. After CLP procedure, WT and CETPTg mice were monitored during 10 days (4 times in the course of daytime) to assess survival. A second set of WT and CETPTg mice was used to sample blood before CLP (0h), and sacrificed 6h or 24h after CLP to sample blood, bile and organs.

CETP activity

CETP activity was measured in plasma with commercial assay kit (Roar Biomedical) according to the manufacturer's instructions. Briefly, plasma was incubated with quenched donor particles and acceptor particles at 37°C. The increase of fluorescence of the reactive mix is related to CETP activity. Cholesteryl ester transfer activity was calculated from the slope of fluorescence increase between 0 and 30 min of incubation. Blank values obtained from WT plasma were subtracted from values obtained in CETPTg plasma.

Plasma parameters

Total cholesterol, free cholesterol, triglycerides, phospholipids, glycaemia and Alanine Transaminase activity (ALT) were measured in plasma and/or lipoprotein fractions using commercially available enzymatic kits on an Indiko automated system (ThermoFisher). Esterified cholesterol levels were calculated by subtracting free cholesterol from total cholesterol values.

FPLC analysis of plasma lipoproteins

Pooled plasma samples (200 µl, seven mice per group) were injected on a Superose 6 HR 10/30 column (GE Healthcare) that was connected to a fast protein liquid chromatography system (Amersham Biosciences). Lipoproteins were eluted at a constant 0.3 mL/min flow rate with 50mM Tris-buffered saline containing 1mM EDTA and 0.02% sodium azide. Cholesterol, phospholipid and protein concentrations were assayed in individual, 0.3-mL fractions. VLDL, LDL and HDL were contained in fractions 7-11, 12-22 and 23-36, respectively.

Cytokine and chemokine assay

Concentrations of interleukin 1 β (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interferon γ (IFNγ), tumor necrosis factor α (TNFα) macrophage chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) in plasma or culture media were determined using a Milliplex MAP Mouse Cytokine / Chemokine Magnetic Bead Panel kit (EMD Millipore), according to the manufacturer's instructions. Samples were analyzed with a Bio-Plex 200 device (Bio-Rad) using Luminex xMAP technology.

Isolation and treatment of bone marrow-derived macrophages (BMDM)

Bone Marrow Derived Macrophages (BMDM) were harvested from CETPTg and WT mice. Rear leg bones (tibia and femur) were flushed using PBS containing 2 mM EDTA and 1% penicillinstreptomycin, and filtered using a 70 µm cell strainer. Red blood cells were lysed (NH4Cl 150 mM, KHCO3 1 m M, EDTA 100 mM) before culture in endotoxin-free RPMI 1640 with L-glutamine containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 50 ng/mL macrophage colony-stimulating factor (M-CSF, Miltenyi Biotec). Cells were plated at 4.0x10 5 cells/mL in 12-well plates (5% CO2, 37°C) during 7 days, with medium replaced every 48 hours. After differentiation, cells were treated during 2, 6, or 24 hours with 10, 100 or 1000 ng/mL LPS (Escherichia coli O55:B5, Sigma Aldrich) in RPMI 1640 with Lglutamine supplemented with 10% pooled plasma from CETPTg or WT mice.

RNA isolation and quantitative PCR

RNAs were isolated from liver homogenates with TRIzol Reagent (Life Technologies) followed by chloroform and isopropanol extraction and quantified with a NanoDrop system (Thermo Fisher Scientific).

Five hundred nanograms of RNAwas were reverse-transcribed into cDNA using M-MLV reverse transcriptase, random primers and RNAseOUT inhibitor (Invitrogen). RNAs were isolated from cells using RNeasy® Mini Kit (Qiagen) and retrotranscripted with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturers' instructions. Quantitative PCR was performed using

StepOne Plus™ Real Time PCR System (Applied Biosystems) with TaqMan Universal Master Mix II (Thermo Fisher) and the following TaqMan probes (Applied Biosystems): IL1beta, Mm00434228_m1; IL6, Mm00446190_m1; IL10, Mm01288386_m1; Mcp1, Mm00441242_m1; Tnfalpha Mm00443258_m1.

Housekeeping gen was Rplp0, Mm00725448_s1. All results were expressed as fold-increase compared with untreated condition (no LPS).

Leukocyte counts in biological samples

Fresh blood samples drawn from retroorbital plexus (10µL) at baseline and 2 hours after LPS injection. At indicated times after CLP, mice were euthanized and submitted to peritoneal lavage with 5 mL PBS. Samples were immediately analyzed on a "Scil Vet abc Plus+" automatic device (scil Animal Care, Altorf, France) in order to obtain total leukocyte (WBC, white blood cells) lymphocyte, monocyte, and polymorphonuclear leukocyte (PMNL) counts.

LPS mass quantitation

LPS concentration in samples was determined by direct quantitation of 3β-hydroxymyristate, a major component of the Lipid A moiety, by liquid chromatography coupled with tandem mass spectrometry (LCMS 2 ) as previously described [START_REF] Pais De Barros | Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[END_REF]. Briefly, an internal standard (3β-hydroxytridecanoate) was added to samples before hydrolysis with HCl for 4h at 90°C. The resulting free fatty acids were extracted with a hexane / ethyl acetate solution and redissolved in absolute ethanol after vacuum evaporation. Fatty acids were separated by HPLC using a Poroshell 120 SB-C18 column and a 1260 Infinity LC system (Agilent Technologies), and quantitated with a 6490 Triple Quadrupole mass spectrometer (Agilent Technologies).

Statistical analysis

Data are presented as means ± SD. and were analysed using GraphPad Prism v6.01 (GraphPad Software Inc.). Wilcoxon rank test was used to compare matched experimental groups within repeated, independent experiments for kinetics of LPS transfer activity in vitro. Survival rates were compared using Kaplan-Meier method and the statistical significance was determined by log-rank test or χ 2 test for specific time points. Correlations were analyzed using non-parametric Spearman test. The other experiments were analyzed using a non-parametric Mann-Whitney test for pairwise comparison. When more than two conditions were considered non-parametric Kruskal-Wallis test followed by Dunn's test was used for multiple comparisons. All statistical analyses are considered as significant with a P-value ≤ 0.05.

RESULTS:

CETP does not transfer LPS in vitro

We set up specific fluorescent assays for quantitation of LPS transfer activity. To this end, purified LPS from Salmonella Minnesota, labelled with a probe bearing a BODIPY dye, was associated with HDL or VLDL donors, and the course of its transfer towards plasma lipoprotein acceptors was monitored over a 30-min incubation at 37°C. As shown in Figure 1, labelled LPS transferred from HDL (Fig. 1A) or from VLDL (Fig. 1B) towards endogenous lipoproteins in a time-dependent manner in plasma of wild-type mice, whereas no significant transfer occurred when donor lipoproteins were incubated alone. The amount of LPS transferred from HDL progressively increased over the 30-min period (Fig. 1A). With VLDL, and for the highest transfer rates, the transfer reached a plateau value between 20 and 30 minutes of incubation (Fig. 1B). As expected from earlier studies (5), the overall transfer of LPS from HDL in PLTP-KO plasma was 19% lower than that in WT plasma after 30 minutes resulting in a 14.6% reduction in AUC of total transfer compared with WT (P<0.05). Initial LPS transfer values from VLDL were significantly lower in PLTP-KO mice, although reaching a similar level after 30 minutes. This resulted in a 26.7% reduction in the AUC of total transfer over 30 minutes compared with WT mice (P<0.05). In accordance with earlier reports, it indicates that plasma PLTP significantly contributes to LPS binding and transfer between lipoproteins in plasma. Importantly, expression of human CETP in CETPTg mice did not modify plasma LPS transfer rates which did not differ to those measured in WT mice with naturally no CETP expression (Fig. 1A and1B). It indicates that CETP, in contrast to the related PLTP, is unlikely to play a significant and direct role in the binding and exchange of LPS between plasma lipoproteins.

The presence of CETP in mice alters the kinetics of purified LPS and its biological effects in vivo

In order to determine whether CETP can alter the clearance of endotoxins in vivo, and the consequences on cytokine production, purified LPS was iv injected in WT and CETPTg mice. LPS concentration in plasma was monitored by using a direct mass assay using the LCMS2 method previously described [START_REF] Pais De Barros | Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[END_REF]. We could observe a faster catabolic rate of LPS in WT than in CETPTg mice with significantly lower LPS levels 3 hours after injection (1.04±0.27 versus 1.58±0.08 µg/mL, respectively, P<0.05, Mann-Whitney) suggesting that detoxification of LPS is slower when CETP is expressed (Fig. 2).

We observed a trend towards a more pronounced rise of plasma IL-6 (6.40 ± 1.75 versus 2.52 ± 0.97 ng/mL) (Fig. 3A, 30 min), IL-10 (10.61 ± 3.42 versus 4.19 ± 1.83 ng/mL) (Fig. 3B, 2 hours) and MIP-2 (84.49 ± 3.63 versus 78.05 ± 5.76 ng/mL) (Fig. 3C, 2 hours) in CETPTg animals at early time points, as well as a similar trend for IL-1β, MCP-1 and IFNγ (Fig. 3 D, E, andF). Hepatic mRNA levels of IL-1β were unchanged in CETPTg mice compared to WT mice after 24 hours (Fig. 3H). Taken together, these data suggest that the delayed clearance of purified LPS transiently alters the cytokine and chemokine burst when CETP is expressed. In order to determine whether CETP exerts its immunomodulatory effect when present in plasma or in innate immune cells we tested the ex-vivo cytokine/chemokine release from bone-marrow derived macrophages (BMDM) from WT or CETPTg mice treated with increasing amounts of LPS in the presence of plasma from WT or CETPTg mice at different time points. As shown in Figure 4, immune response was similar between macrophages from WT and CETPTg mice. However, cytokine production was significantly altered in the presence of plasma from CETPTg mice compared with plasma from WT mice. Indeed, in the presence of CETPTg plasma, when compared with WT plasma, IL-6 levels in culture media was decreased at 6 hours with 1000 ng/mL LPS (-42.2%, Fig. 4A); MCP-1 was increased at 24h for all doses of LPS tested (+54.6%, +95.4% and +46.7%, for 10, 100 and 1000ng/mL, respectively, Fig. 4B); IL-10 was increased when treated for 24 hours with 10 mg/mL LPS (+53.3%, Fig. 4C); TNFα was increased at 2h when treated with 10 and 100 ng/mL LPS (+34% and +37.3%, respectively, Fig. 4D); and IL-1β was increased at 6 hours when treated with 100 and 1000 ng/mL LPS (27.0-fold and 6.4-fold increases, respectively Fig. 4E) (P<0.05 in all cases, Kruskal-Wallis followed by Dunn's multiple comparison test).To get more insight into the possible impact of CETP on mRNA levels of inflammatory mediators, macrophages were isolated from bone marrow from WT an CETPTg mice and submitted to LPS treatment (100 ng/mL, 6 hours) in the presence of plasma from WT or CETPTg mice. As shown in Figure S1, neither CETP expression in macrophages nor the presence of CETP in plasma induced changes in cytokine mRNA levels.

Finally, in order to assess the impact of CETP on metabolic parameters and survival during endotoxemia, we challenged WT and CETPTg mice with a lethal dose of LPS (15mg/kg) delivered iv. The presence of CETP in transgenic mice did not alter plasma cytokine levels two hours after LPS injection (Fig. S2). Leukocyte counts were identical at baseline and decreased after LPS injection, as expected from previous studies [START_REF] Gautier | Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice[END_REF][START_REF] Steven | Time Response of Oxidative/Nitrosative Stress and Inflammation in LPS-Induced Endotoxaemia-A Comparative Study of Mice and Rats[END_REF][START_REF] Kamochi | P-selectin and ICAM-1 mediate endotoxin-induced neutrophil recruitment and injury to the lung and liver[END_REF]. The extent of this decrease was similar in WT and CETPTg mice (Fig. S3).

However, total and esterified plasma cholesterol levels, which are known to be altered in CETPTg mice under basal conditions [START_REF] Hayek | An interaction between the human cholesteryl ester transfer protein (CETP) and apolipoprotein A-I genes in transgenic mice results in a profound CETP-mediated depression of high density lipoprotein cholesterol levels[END_REF], remained lower one hour after LPS injection compared to WT counterparts (0.81 g/L ± 0.14 versus 1.01 g/L ± 0.14, and 0.73 g/L ± 0.13 versus 0.89 g/L ± 0.10, respectively; P<0.05) (Fig. 5A,B). Plasma phospholipid levels were also found to be lower in CETPTg mice 1 hour after LPS injection (1.81 g/L ± 0.21 versus 2.08 g/L ± 0.24; P<0.05) (Fig. 5C) while triglyceride levels were similar in both groups (Fig. 5D). In order to get more insights into the impact of CETP expression on lipoprotein profile during severe endotoxemia, pooled plasmas obtained from WT and CETPTg mice at baseline and two hours after LPS injection (15mg/kg) were passed through a size exclusion chromatography device and cholesterol, phospholipid, triglyceride and protein concentrations were determined in individual fractions.

As expected in mice, HDL appeared as the main carriers for cholesterol (more than 75%, Fig. 5E) and phospholipids (more than 80%, Fig. 5F) in plasma, both in WT and CETPTg mice. The HDL peak was smaller and shifted to a lower size range in CETPTg mice compared with WT mice at baseline and after LPS injection (Figure 5E,F). In parallel, we also measured protein content in all FPLC fractions. At baseline, cholesterol to protein ratio in HDL was 15% lower in CETPTg mice compared to WT mice. LPS injection did not induce any other change in cholesterol to protein ratio in HDL from both genotypes and an 11% reduction in this parameter was still observable in CETPTg mice. A similar trend could be observed in LDL (-17% at t=0; -8% at t=2h) and in VLDL (-55% at t=0; -41% at t=2h) when comparing CETPTg mice to WT mice. Phospholipid to protein ratios followed the same trend in HDL (-17% at t=0; -9% at t=2 hours), LDL (-16% at t=0 ; -31% at t=2h) and VLDL (39% at t=0h ; -53% at t=2h) in CETPTg mice compared with WT mice. Regarding triglyceride-rich lipoproteins (VLDL), CETPTg mice displayed a 48% drop in TG to protein ratio at baseline and this trait was still observed two hours after LPS injection (-37%).

After a 10-day period of follow-up, mortality rate was 50% higher in CETPTg mice than in WT counterparts (90% versus 60%, respectively; P<0.05, log Rank test) (Fig. 6A). In order to evaluate whether higher mortality in mice might be linked to the amount of circulating lipids or cytokines, survival time after LPS injection was plotted against plasma concentrations of total lipids. A significant, positive correlation could be observed between survival time and plasma lipids (r 2 = 0.3565 ; P = 0.0146, Spearman test, Figure 6B).

Effect of CETP on LPS detoxification and sepsis outcome in a CLP model

As part of the outer membrane of gram-negative bacteria, most cases of endotoxemia does not consist in the presence of isolated LPS molecules only but rather in bacterial blebs or complex hydrophobic structures released during sepsis. In order to get more insights into the possible role of CETP on LPS detoxification and its consequences on sepsis outcome in a model of polymicrobial infection, we applied the CLP protocol to CETPTg and WT mice. In both WT and CETPTg mice, sepsis triggered a rapid and persistent elevation of LPS mass concentration in plasma, with similar observations whether CETP was expressed or not (Fig. 7A). However, while WT mice displayed significant increase in biliary excretion of LPS after 24 hours (168 ± 79 ng/mL at 24h versus 24 ± 7 ng/mL at baseline, P<0.05), the presence of CETP tended to limit LPS excretion with no significant accumulation in bile at 24h compared with baseline (Fig. 7B). The LPS content of the liver was similar between 6h and 24h of sepsis in WT and CETPTg mice (Fig. 7C).

Next, we checked the possible effect of CETP on metabolic parameters in our CLP model. Plasma lipid parameters were measured in WT and CETPTg mice at baseline (0h, Table 1) and 6h and 24h after application of the CLP procedure (Table 2). At baseline, and as expected [START_REF] Gautier | Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice[END_REF], total and esterified cholesterol levels were lower in CETPTg mice as compared to WT mice (0.78 g/L ± 0.15 versus 0.92 g/L ± 0.20, and 0.46 g/L ± 0.09 versus 0.62 g/L ± 0.12, respectively; P<0.05) (Table 1). CETPTg mice showed higher apoBcontaining lipoproteins to HDL cholesteryl ester ratio as compared to WT mice (Fig. S4A). It is in line with expected outcome of CETP expression, and CETP activity remained stable along the sepsis episode (Fig S4B). Total cholesterol, esterified cholesterol and phospholipid levels increased significantly between 6h and 24h after CLP in wild-type mice only (Table 2). The same pattern was observed for ALT activity.

The induction of sepsis originating from the peritoneal cavity induced the accumulation of leukocytes (WBC) in both WT and CETPT mice (Fig. S5A). This accumulation did not relate to alterations in lymphocyte counts (Fig. S5B) and only marginally to alterations in monocytes (Fig. S5C). Rather, leukocyte accumulation was mostly due to recruitment of polymorphonuclear neutrophils (PMNL), (Fig. S5D). Interestingly total WBC and PMNL progressively increased until 24h in WT mice while they reached a peak at 6h before going down at 24h in CETPTg mice (Fig. S5A andS5D). To investigate further whether CETP could affect systemic inflammatory response induced by sepsis, plasma levels of cytokines and chemokines were monitored over a 24-hour period after CLP. Although the progressive rise in plasma levels of IL-6, IL-10, MCP-1 and MIP-2 after CLP was not affected by CETP expression (Fig. 8A,B,C,D), increases in plasma levels of IL-1β, TNFα, and IFNγ after 24h hours were observed in CETPTg mice only (Fig. 8E,F,G).

Finally, the consequences of CETP expression on mouse survival after polymicrobial infection and endotoxemia were determined. A significantly higher rate of mortality could be observed in the early days in CETPTg than in WT mice, in particular with 50% versus 10% of mortality in CETPTg and WT mice after 3 days of sepsis, respectively (P<0.05, χ 2 test). Ten days after CLP, both WT and CETPTg groups displayed 75% of mortality (Fig. 9).

DISCUSSION:

Although CETP belongs to the LT/LBP family, which includes members with well-described LPS transfer activities, the role of CETP in LPS transfer remains unclear. It is reported here that, unlike LBP, BPI and PLTP, CETP has no intrinsic LPS transfer activity, with consistent in vitro and in vivo observations. Importantly however, we could show that CETP expression was associated with a change in inflammatory burst and with earlier or higher mortality, both in polymicrobial infection and acute endotoxemia models.

The first part of this work aimed at clarifying the role of CETP in LPS binding and transfer activity.

Earlier in vitro works showed that CETP seems to be partially co-localized with LPS in the perinuclear region of macrophages [START_REF] Venancio | CETP Lowers TLR4 Expression Which Attenuates the Inflammatory Response Induced by LPS and Polymicrobial Sepsis[END_REF], suggesting a possible interaction between these molecules; CETP was also found to enhance LPS binding to purified HDL and LDL at the expense of VLDL [START_REF] Cazita | Human cholesteryl ester transfer protein expression enhances the mouse survival rate in an experimental systemic inflammation model: a novel role for CETP[END_REF]. However, these latter results could not be reproduced in vivo since we observed no increase in lipoprotein-bound LPS in CETPTg mice compared to wild-type animals in our model of polymicrobial infection. One of the drawbacks of the technique used in earlier in vitro studies comes from the use of purified LPS as a substrate for CETP [START_REF] Cazita | Human cholesteryl ester transfer protein expression enhances the mouse survival rate in an experimental systemic inflammation model: a novel role for CETP[END_REF] while circulating LPS is mostly found in host organisms as aggregates/micelles or as part of complex lipid structures derived from bacterial membranes during sepsis [START_REF] Gegner | Lipopolysaccharide (LPS) Signal Transduction and Clearance: Dual Roles for LPS Binding Protein and Membrane CD14[END_REF]. In order to get more insight in the LPS transfer potency activity of CETP, we developed here a novel technique that allows us to monitor the net transfer of LPS from donors (HDL or VLDL) to acceptor lipoproteins. In addition, our transfer systems used total plasma as a source of endogenous lipid transfer proteins and lipoprotein acceptors, thus reflecting more accurately what may occur in vivo. Our technique could be validated by observing a significant decrease in LPS transfer activity in plasma from knockout mice lacking PLTP, i.e. one of the proteins known to bind and transfer LPS in vitro and in vivo, compared to plasma from WT mice with naturally elevated plasma PLTP activity. Consistent observations were made using either HDL or VLDL as donors. In the presence of plasma from CETPTg mice, LPS transfer from HDL or VLDL to endogenous lipoproteins was not modified compared to plasma from WT mice lacking CETP. The absence of LPS transfer activity of CETP contrasts with two other circulating members of the LT/LBP family, LBP and PLTP, which both bind and transfer LPS to lipoproteins. Taken together with the work of Clark and colleagues, who showed that CETP has a very low affinity for LPS (Kd ≥ 25 µM) [START_REF] Clark | Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection[END_REF], our data suggest that 1) CETP has no direct interaction with LPS, and 2) does not promote its transfer to and between lipoproteins. Therefore, the deleterious effect of CETP that we could observe in both models of endotoxemia and polymicrobial infection might relate to an indirect role of this protein on sepsis and endotoxemia, independently from LPS transfer.

Lipoproteins may play a pivotal role in the resistance against sepsis through different mechanisms.

Firstly, HDL harbor intrinsic anti-inflammatory properties by decreasing the expression of adhesion molecules in the vascular wall, the migration of immune cells or the production of cytokines and chemokines [START_REF] Arora | HDL-A molecule with a multi-faceted role in coronary artery disease[END_REF]. Secondly, while HDL were shown to promote LPS transport back to the liver for biliary excretion [START_REF] Gautier | Plasma PLTP (phospholipid-transfer protein): an emerging role in "reverse lipopolysaccharide transport" and innate immunity[END_REF][START_REF] Van Lenten | The role of lipoproteins and receptor-mediated endocytosis in the transport of bacterial lipopolysaccharide[END_REF][START_REF] Munford | Binding of Salmonella typhimurium lipopolysaccharides to rat high-density lipoproteins[END_REF], all lipoprotein classes are able to bind and inactivate LPS molecules that are present in lymph and plasma [START_REF] Levels | Lipid composition and lipopolysaccharide binding capacity of lipoproteins in plasma and lymph of patients with systemic inflammatory response syndrome and multiple organ failure[END_REF]. Due to its ability to exchange neutral lipids (CE and TG) between lipoproteins, CETP induces profound changes in the composition, functionality and metabolic fate of lipoproteins, including decreased HDL cholesterol levels and HDL size in humans [START_REF] Kuivenhoven | Heterogeneity at the CETP Gene Locus : Influence on Plasma CETP Concentrations and HDL Cholesterol Levels[END_REF] and animal models [START_REF] Whitlock | Monoclonal antibody inhibition of cholesteryl ester transfer protein activity in the rabbit. Effects on lipoprotein composition and high density lipoprotein cholesteryl ester metabolism[END_REF][START_REF] Agellon | Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice[END_REF].

In accordance with a modulation of LPS clearance from plasma, we could show that the presence of CETP could delay the clearance of endotoxins after a single iv injection of LPS. This observation could be explained by the propensity of CETP to modulate the ability of HDL to promote the clearance of circulating lipids by the liver [START_REF] Collet | Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I[END_REF][START_REF] Botham | The hepatic uptake of rat high-density lipoprotein cholesteryl ester is delayed after treatment with cholesteryl ester transfer protein[END_REF][START_REF] Catalano | Torcetrapib differentially modulates the biological activities of HDL2 and HDL3 particles in the reverse cholesterol transport pathway[END_REF]. In addition, we could show a slight alteration of LPS biliary output in CETPTg mice compared with WT mice under CLP. The lack of apparent effect of CETP on the time-course of plasma LPS levels in our sepsis model might relate to the peculiar kinetic behavior of LPS which is progressively and constantly delivered into the circulation over at least 24 hours after CLP (11, 37, 38, present study), contrarily to our endotoxemia model which consists in an unique LPS bolus.

In clinics, a low cholesterol level at the day of admission is a poor prognostic of survival in patients suffering septic shock [START_REF] Chien | Low serum level of high-density lipoprotein cholesterol is a poor prognostic factor for severe sepsis[END_REF][START_REF] Memiş | High Creactive protein and low cholesterol levels are prognostic markers of survival in severe sepsis[END_REF] and is associated with more severe inflammatory burst and sepsis outcome in patients undergoing surgery under cardiopulmonary bypass [START_REF] Lagrost | Low Preoperative Cholesterol Level Is a Risk Factor of Sepsis and Poor Clinical Outcome in Patients Undergoing Cardiac Surgery With Cardiopulmonary Bypass[END_REF]. It indicates that the initial level of cholesterol is a critical parameter in sepsis outcome and inflammatory response. Recent work showed that a gain-of-function variant in CETP gene was associated with lower plasma HDL cholesterol levels, more pronounced pro-inflammatory cytokines release, and greater risk of sepsis-associated acute kidney injury [START_REF] Genga | CETP genetic variant rs1800777 (allele A) is associated with abnormally low HDL-C levels and increased risk of AKI during sepsis[END_REF]. CETP-mediated HDL-C drop in septic patients was later shown to play a causal and deleterious role in survival from sepsis [START_REF] Trinder | Cholesteryl Ester Transfer Protein Influences High-Density Lipoprotein Levels and Survival in Sepsis[END_REF]. Hence, basal changes in plasma total cholesterol and lipoprotein levels, as occurring in the presence of CETP, may significantly alter inflammation and sepsis outcome. Interestingly, under a chow diet, CETP expression in transgenic mice is also associated with decreased total cholesterol levels when compared to wild-type mice devoid of CETP [START_REF] Gautier | Human apolipoprotein C-I accounts for the ability of plasma high density lipoproteins to inhibit the cholesteryl ester transfer protein activity[END_REF][START_REF] Melchior | Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into the mechanisms responsible for low plasma high density lipoprotein levels[END_REF]. The present study showed significant reductions in plasma levels of total and esterified cholesterol at the basal state and early time points in CETPTg mice compared to wild-type mice as well as significant decreases in plasma phospholipids in CETPTg mice compared with WT mice after LPS injection or during CLP. This consistent CETP-related decrease in plasma lipids in both models might relate to an enhancement of the remodeling of lipoproteins that is observed either at baseline or during inflammation [START_REF] Levels | Alterations in lipoprotein homeostasis during human experimental endotoxemia and clinical sepsis[END_REF]. In support of this hypothesis, CETP was shown to favor PLTP-induced changes in HDL structure and composition in vitro [START_REF] Rye | Triglyceride-enrichment of high density lipoproteins enhances their remodelling by phospholipid transfer protein[END_REF]. Accordingly, in the present study FPLC analysis showed that the reduction in HDL size and lipid content in the presence of CETP tended to be further pronounced during endotoxemia. Several studies indicated that phospholipids reflect the total amount of lipoprotein surface as well as their capacity to bind and/or inactivate LPS while cholesterol and triglycerides are mostly linked with the content of lipoprotein core [START_REF] Cué | Reconstituted high density lipoprotein inhibits physiologic and tumor necrosis factor alpha responses to lipopolysaccharide in rabbits[END_REF][START_REF] Parker | Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood[END_REF][START_REF] Kitchens | Acute inflammation and infection maintain circulating phospholipid levels and enhance lipopolysaccharide binding to plasma lipoproteins[END_REF]. Therefore we considered that the sum of total plasma lipids (cholesterol, triglycerides and phospholipids) could reflect the overall capacity of plasma lipoproteins to counteract the deleterious effects of endotoxemia and we could show that there was indeed a significant, positive correlation between total lipid values and survival rate in mice injected with LPS. In addition, we could also show ex vivo that plasma from CETPTg mice is able to modulate LPS-induced cytokine secretion from macrophages when compared with plasma from WT mice.

The latter observation suggests that, beyond its influence on LPS kinetics, alterations of plasma profile in the of CETP directly impact the pro-inflammatory effect of LPS. Taken together, these data provide evidence that increased mortality and altered inflammatory response in CETPTg mice with endotoxemia or sepsis result from a general reduction in plasma lipid pool as a result from increased CETPmediated lipoprotein remodeling and/or clearance.

Overall, whereas the present study comes in support of a significant role of CETP in modulating LPS clearance and LPS-mediated inflammation, its actual role and consequences must be evaluated and discussed in conjunction with other key players including immune cells, lipoproteins and other LPS binding proteins. In addition, the outcome of CETP and lipoprotein actions might well depend on the time-course of the inflammatory process. For instance, although HDL are mostly considered as anti-inflammatory, they were shown to behave as pro-inflammatory factors in the early step of inflammation due to their ability to either unmask LBP or to deplete macrophage cholesterol [START_REF] Pirillo | HDL in infectious diseases and sepsis[END_REF][START_REF] Thompson | Native high-density lipoprotein augments monocyte responses to lipopolysaccharide (LPS) by suppressing the inhibitory activity of LPS-binding protein[END_REF][START_REF] Van Der Vorst | High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling[END_REF]. Interestingly, Blauw and colleagues have recently discussed the primacy of this early and transient pro-inflammatory effect of HDL to trigger a subsequent immune response required for sepsis resolution [START_REF] Blauw | A Novel Role for CETP as Immunological Gatekeeper: Raising HDL to Cure Sepsis? Trends[END_REF]. As far as CETP is concerned, and beyond the intravascular LPS metabolism that was under the scope of the present study, it was found to modulate the expression of TLR-4 receptors [START_REF] Venancio | CETP Lowers TLR4 Expression Which Attenuates the Inflammatory Response Induced by LPS and Polymicrobial Sepsis[END_REF]. Finally, the importance the IL-6 / IL-10 balance was stressed in predicting the sepsis outcome and mortality [START_REF] Chaudhry | Role of cytokines as a double-edged sword in sepsis[END_REF]. Because both IL-6 and IL-10 were found in the present study to be increased in CETPTg mice, the consequences of CETP on clinical outcome in relation with cytokine expression should be considered with caution and may not be restricted to sepsis and LPS clearance. Interestingly, total WBCs, mostly reflecting PMNL recruitment in the peritoneal cavity of mice subjected to CLP showed a different pattern: while the recruitment was sustained until 24h hours in WT mice, PMNL counts in CETPTg mice were transiently observed at 6h hours only. These differences in the kinetics of PMNL recruitment in the peritoneal cavity might partly contribute to the differences in survival between CETPTg and WT mice after CLP.

In conclusion, our observations showed that CETP has no LPS binding or transfer capabilities in vitro and thus appears as an outlier in the LT/LBP family. Nevertheless, it is still able to influence the inflammatory response through its ability to alter lipoprotein levels, kinetics and functionality. Although we could show that CETP is able to alter LPS transport and excretion in a model of isolated endotoxemia, the contribution of this phenomenon to the deleterious effect of CETP in our sepsis model seems to be modest.

Rather, the lowering effect of CETP on lipid levels combined with altered intrinsic anti-inflammatory capacity of plasma lipoproteins, might contribute to clinical outcome. WT (n=4) and CETPTg (n=4) mice were iv injected with LPS (1.0 mg/kg BW). LPS concentration in plasma was monitored at 0 min, 30 min, 1h, 3h, 6h and 24h by direct 3-hydroxymyristate (3-HM) quantitation using tandem mass spectroscopy. Data are means ± SD. * P < 0.05 WT vs. CETPTg; Mann-Whitney test.

Figure 1 :Figure 2 :

 12 Figure 1: Role of CETP in LPS transfer activity between lipoproteins.

*Figure 3 :

 3 Figure 3: Cytokine production after LPS injection.

Figure 4 :

 4 Figure 4: Cytokine release in the presence of plasma and BMDM from WT and CETPTg mice

Figure 5 :Figure 6 :

 56 Figure 5: Plasma parameters and lipoprotein profile in mice after LPS iv injection. WT (n=13) and CETPTg (n=12) mice were iv injected with purified LPS from E. coli O55:B5 (15.0 mg/kg

*Figure 7 :Figure 8 :

 78 Figure 7: LPS distribution and excretion after CLP.

Figure 9 :

 9 Figure 9: Survival after CLP.

TABLES : Table 1 : Plasma parameters in mice at baseline.

 :1 Lipid parameters, glycemia and liver injury marker alanine aminotransferase (ALT) were measured at baseline in plasma of WT (n=6 to 8) and CETPTg (n=6 to 8) mice. Data are means ± SD. *P<0.05 vs. WT, Mann-Whitney test.

Table 2 : Plasma parameters in mice after CLP.

 2 WT (n=6 to 8) and CETPTg (n=6 to 8) mice were challenged with CLP. Lipid parameters, glycemia and liver injury marker alanine aminotransferase (ALT) were measured in plasma 6h and 24 h after CLP. Data are means ± SD. # P<0.05, ## P<0.01 vs. t6h; Kruskal-Wallis test with Dunn's multiple comparisons test.

	Baseline
	WT	CETPTg

ACKNOWLEDGMENTS / GRANT SUPPORT:

This work was supported by grants from the University of Bourgogne Franche-Comté, the Institut National de la Santé et de la Recherche Médicale (INSERM), the Fondation de France (Grant Project Cardio 2013 R13057MM, reference RAF13002MMA), by ANR grant ATHEROLIP and by a French Government grant managed by the French National Research Agency under the program 'Investissements d'Avenir' with reference ANR-11-LABX-0021 (LipSTIC Labex).


Keywords: Metal (IV) Metal-organic frameworks, Room temperature synthesis, Defective MOF, Scale-up synthesis

The development of room temperature green syntheses of robust MOFs is of great interest to meet the demand of the sustainable chemistry and is a pre-requisite for the incorporation of functional but fragile compounds in water stable MOFs. However, only few ambient conditions routes to produce metal(IV) based MOFs have been reported and most of them suffer from a very low yield and/or multiple steps that preclude their use for most applications. We report here a new versatile one-step synthesis of a series of highly porous M6 oxoclusters based MOFs (M= Zr, Hf, Ce) at room temperature, including 8 or 12-connected micro/mesoporous solids with different functionalized organic ligands. The resulting compounds show varying degrees of defectivity, particularly for 12connected phases, while maintaining the chemical stability of the parent MOFs. We propose first insights for the efficient MOF preparation based on In-situ kinetics observations. Remarkably, the synthetic versatility not only allows an efficient room temperature synthesis with a high space-time yield, but also gives possibility to tune the particle size, which therefore paves the way for their practical use.

Introduction

Room temperature (RT) green-synthesis of Metal-organic frameworks (MOFs) have generated interest in last two decades. [1] In addition to the interest of industry for lower energy cost and safer conditions, the ambient conditions synthesis could also lead to some property enhancements (e.g. catalysis, gas adsorption) including the use of fragile compounds. [2] Most low temperature routes reported to date deal with divalent metals based carboxylate MOFs. However, these solids strongly suffer from their poor chemical stability, particularly upon exposure to moisture, which prevents their practical use. [3] With the more chemically robust Zeolitic Imidazolate Frameworks (ZIFs), low temperature synthesis allows however to construct core-shell materials through the direct formation of ZIFs in the presence of pre-formed functional molecules or nanoparticles in a view of several potential applications (sensing, catalysis, biomedicine…). [4] Additionally, it is a convenient method to introduce low temperature-induced defects (LTID), as illustrated recently with the RT synthesis of ZIF-67 that led to the improvement of the catalytic activity for the synthesis of cyclic carbonates. It also allows using techniques that requires ambient conditions, such as the investigation of the MOF nucleation-growth kinetics at RT using in-situ Transmission-Electron-Microscope coupled with a liquid cell. [5] Trivalent metals (e.g. Fe, Al, Cr) based carboxylate MOFs are generally more chemically robust than the aforementioned divalent ones, particularly with Al(III) and Cr(III). [6] In such case, the chemistry in play is more complex and developing versatile and easy room temperature synthesis routes is an old but still highly challenging objective. [7] For example, despite efforts devoted to the sustainable synthesis of Trivalent-MOFs from the MIL family (MIL stands for Materials from Institut Lavoisier), only few of them can be so far synthesized using green solvents at room temperature, including MIL-53(Al), MIL-88A(Fe) and MIL-100(Fe). [8] Zr(IV) carboxylate based MOFs are considered as one of the most promising sub-class of MOFs due to their low toxicity and high chemical stability, making them appealing candidates for applications such as heterogeneous catalysis, gas/liquid separation and bio-applications. [9] These MOFs are typically synthesized using solvothermal conditions with toxic solvents such as DMF. Alternatively, one can synthesize these solids at ambient pressure using conventional [10] or microwave-assisted heating. [11] In 2010, some of us reported a room temperature synthesis of UiO-66 type MOFs, starting from preformed Zr6 oxoclusters and dicarboxylic acids in DMF. [12] Since this pioneer study, the reaction of preformed Zr6/ Zr12 oxoclusters with carboxylic acid linkers at room temperature using both solvent and solvent-free reactions has been explored. [13] However, this strategy relies on the preparation of non-commercially available oxoclusters and in some cases gives rise to poorly crystalline solids with a relatively low yield. Despite the complexity of this RT method, it has been used to incorporate thermally sensitive compounds into Zr-MOFs. For example, Farha et al. reported the RT preparation of the 8-connected Zr-MOF NU-901 via a two steps approach, allowing the formation of core-shell Pd Nanorods@NU-901 for selective surface-enhanced Raman spectroscopy. [14] RT routes to prepare metal(IV) MOFs is also of interest to produce LTID and tune their properties, including porosity, Lewis acidity and hydrophilicity. [15] Few recent reports describe one-step RT synthetic approaches of UiO-type MOFs. [8b, 16] However, they rely on specific reactants or additives to promote nucleation, associated to cost issues and/or to the presence of impurities in the resulting products. Therefore, despite promises, there is still a strong need to develop a versatile simple higher yield and environmentally friendly RT approach to produce robust metal(IV) MOFs.

Here, we report a new more efficient one-step environmentally friendly route for the synthesis of metal(IV)-based MOFs at room temperature, including archetypical MOFs based either on 12connected (MOF-801, UiO-66-NH2, UiO-66-COOH) and 8connected (DUT-67, PCN-222) M6 oxoclusters (M=Zr, Hf, Ce). MOF-801 is used as a representative example in order to demonstrate the broad scope of available synthetic conditions, including the use of organic or inorganic Zr(IV) salts, of several tetravalent metal ions (Zr(IV), Hf(IV) or Ce(IV)) and of different types of modulators. In a second step, the extension of this strategy to several other benchmark MOFs is described. In-situ PXRD technique is finally considered to shed light on the nucleation/growth process, revealing the importance of the concentration on the synthetic efficiency. Finally, we demonstrate how our findings enable the successful laboratory pilot scale production of MOF-801, with a space-time yield (STY) of 168 kg/m 3 /day, among the highest for the RT synthesis of Zr-MOFs. We believe such novel green ambient conditions synthesis routes of Zr(IV)-carboxylate based MOFs could not only be extended to series of benchmark MOFs on a larger scale, paving the way towards their industrial production, but also be applied to combine MOFs with temperature sensitive species to develop new functional MOFs based composites.

Results and Discussion

MOF-801 or Zr-fumarate was selected as a prototypical example for our investigation due to the good solubility of fumaric acid in H2O and the low price of the reactants. It presents a similar structure as UiO-66 with fumaric acid as linker instead of terephthalic acid. As illustrated in Figure 1 (b), each Zr6 oxocluster is coordinated to 12 fumaric acids, giving rise to a cubic 3D structure with two 5.6Å and 4.8Å types of tetrahedral cages and one 7.4Å type of octahedral cage. PXRD patterns evidenced the successful synthesis of well-crystalized MOF-801 with Zr(IV), Hf(IV) and Ce(IV) in water at room temperature using formic acid as modulator (Figure 2(a)). Hf(IV)-based and Ce(IV)based MOF analogues are usually less investigated comparing to Zr(IV) ones, despite their unique advantages in terms of high acidity and/or redox properties. [17] A slight shift of the diffraction peaks was observed for MOF-801(Ce) due to the larger atomic radius of Ce(IV) compared to Zr(IV). Additionally, as formic acid is usually considered as a less favorable inhibitor due to its higher acidic character, we also demonstrated the possibility (see Figure S15) to synthesize MOF-801(Zr) using acetic acid as inhibitor. The successful use of diverse metal salts in this approach, such as ZrOCl2 and Zr(iPrO)4 for the synthesis of MOF-801(Zr) was also demonstrated (Figure S16). This is of interest particularly if one wants to minimize any corrosion or moisture stability issues when using ZrCl4.

N2 adsorptiondesorption isotherms at 77K (Figure 2(d) and Table 1) evidenced that these materials are highly porous, with Brunauer-Emmett- Teller (BET) surface area of 1035(±5) m 2 /g (MOF-801(Zr)), 735(±6) m 2 /g (MOF-801(Hf)) and 780(±5) m 2 /g (MOF-801(Ce)) respectively. These values are slightly larger than the pristine MOF-801(Zr) (680 m 2 /g), which suggests that these MOFs present a significant amount of defects, as reported previously using other synthesis conditions. [18] In order to determine the defect nature (missing nodes or linkers), FT-IR analysis was carried out (Figure S1). All the materials showed only vibrations associated to COO -groups coordinated to Zr(IV), without traces of uncoordinated fumaric acid, suggesting that missing linkers are more plausible than missing nodes. Thermogravimetric analysis (TGA) further provided the evidence of the lower connectivity of the Zr6 nodes compared to the ideal structure.

According to the ratio of decomposed content of ligands and remaining metal oxide calculated from TGA (Table 1, Figure S2(a)-( c)), a connectivity between 7 and 10 is obtained depending on the samples, while a connectivity of 12 is expected for the perfect structure. Finally, in the case of MOF-801(Zr) and MOF-801(Hf), two low-intensity peaks at low angle (marked with blue dash line) appeared, in agreement with the presence of missing-linker defects in the body-centered initial fcu structure. [19] In a second step, we extended our strategy to a broader scope of Zr(IV)-based MOFs with different organic ligands and topologies to highlight the versatility of the approach. As shown in Figure 2 (b, c), PXRD patterns evidenced that highly crystalline UiO-66-NH2, UiO-66-COOH (fcu topology, see structure in Figure 1(c)) and DUT-67(PDA) (reo topology, see structure in Figure 1(d)) were synthesized successfully following a similar synthesis protocol. Notably, synthesizing the functionalized UiO-66-COOH at room temperature is not straightforward due to the lower pKa of Trimellitic acid in comparison to other derivatives (e.g. -NH2, -H) that usually favors the formation of amorphous compounds. [8b, 13c] The room temperature obtained MOFs presented higher surface areas (UiO-66-NH2 (1255(±5) m 2 /g), UiO-66-COOH (1050(±6) m 2 /g) and DUT-67(PDA) (1020(±6) m 2 /g)) than the theoretical values (see Figure 2(e, f)), in agreement with the presence of defects, as mentioned above for MOF-801 (see Table 1). Pore size distribution for UiO-type MOFs (see Figure S10(a), (b)) revealed indeed the defectengineering pore size expansion, showing a wide pore size distribution from 0.6 to 1.6 nm, which is much larger than the one deduced from the crystal structures [20] . In the latter case, DUT-67(PDA) showed a similar 77K N2 adsorption capacity than the reported one obtained on a compound prepared using solvothermal synthesis. This is in agreement with TGA, which indicates that only 0.2 linker per metal nodes are missing in this case. [21] In addition to the use of short dicarboxylic ligands described above, PCN-222, a highly chemically stable MOF based on a 4,8-connected framework (see structure in Figure 1(e)) comprising Zr6 oxoclusters and TCPP (tetrakis(4carboxyphenyl)porphyrin), was synthesized at room temperature. [22] Due to the poor solubility of TCPP in many solvents, including DMF, we used Zr(iPrO)4 (Zirconium(IV) isopropoxide), a more basic zirconium source than ZrCl4 and ZrOCl2 that may enhance the linker solubility via a partial deprotonation of TCPP. Figure 3(a) shows that highly crystalline PCN-222 microcrystals were obtained, confirming that our room temperature synthesis strategy is also suitable for metal(IV) carboxylate MOFs built with large poorly soluble ligands. Even though the product yield (56%) is lower than for the other cases presented in this work, it is still comparable with the reported one using high-temperature synthesis in DMF. [22] Apart from the low solubility of TCPP in EtOH, the contamination of the product during the synthesis by coordination of TCPP to unsaturated Zr sites in the large hexagonal pores of the MOF can further decrease the product yield. Notably, when one considers the preparation MOFs based on polyphenylene ligands and high valence metal ions, mixed phases are often obtained, which was not the case here as confirmed by PXRD and SEM (Figure S11). [23] N2 adsorption-desorption isotherms in Figure 3(b) showed high porosity and a typical shape of isotherm due to the hierarchical pore of PCN-222.

In order to investigate the chemical stability of all these synthesized MOFs, the compounds were soaked in different chemical environments (initial pH= 0, 12, boiling water for 2 days). PXRD measurements in Figure S3-9 proved that all these materials present -as previously reported-a high chemical stability even in presence of a large amount of missing ligands.

The above-mentioned results demonstrated the versatility of this new environmentally friendly synthesis approach. We then investigated the possibility to take benefit from this strategy to produce nanosized MOFs (nanoMOFs). These latter have been extensively studied for their appealing advantages in catalysis, sensing, bio-applications and membrane science. [24] Usually, the particle size tuning was realized following several strategies such as microwave heating, sonication, fluidics, or the use of emulsions, modulators, shorter reaction time or temperature control. [25] In spite of diverse synthetic methods, a scalable green synthesis of nanoMOFs with high product yield is still highly demanded and only few examples have been reported so far. [26] Here, in the case of MOF-801(Zr), decreasing the amount of modulator allowed particle size tuning from ~220 nm to ~45 nm according to SEM, TEM images (Figure S12 and Figure S13) and PXRD (see broadening of the Bragg peaks on Figure S14). Noteworthy, whatever the particle size, a well-crystalline MOF-801(Zr) without any decrease of the yield (89%). In order to prepare Zr-MOFs at ambient temperature without any detrimental effect on the kinetics of nucleation, the lowering of activation energies is needed. Previous reports showed that activation energies of nucleation and growth are close for UiOtype MOFs (71 kJ mol -1 vs 66 kJ mol -1 ). [27] Few factors have been found to lower down these values, including the addition of HCl, [28] water and/or the use of more soluble ligands. [27,29] In our case, the mixture of ZrCl4 (or ZrOCl2) and pure H2O (~300 equiv H2O/ Zr) led to relatively low pH values (≈0.24, experimentally) in comparison to the conventional synthesis in DMF but hydrolysis of Zr 4+ species was favored by the excess of water that led to the easier formation of Zr6 oxoclusters with bridging μ3-O and μ3-OH groups. Thus, this might explain why our conditions favor the formation of Zr6 oxoclusters at room temperature. [30] Moreover, the Arrhenius activation energies for the MOF growth were expected to be relatively low owing to the reasonable solubility of the chosen linkers in H2O/ Ethanol. The temperaturedependent in-situ nucleation experiments reported previously in the presence of H2O, ZrCl4/ ZrOCl2, HCl, and DMF by some of us indicated that lowering down the temperature had a significantly negative impact on the nucleation time of UiO-66, particularly when the temperature was lower than 90°C. [28] In a nut-shell, the nucleation time was increased ca. 2 times when decreasing every 10°C. Accordingly, keeping identical the other synthesis parameters, the nucleation time should have reached almost 40 days at room temperature. In our case, the synthesis time was only of 1-2 days (spinning in a capillary). Thus, apart from the synthetic parameters aforementioned, we hypothesize that the use of lower concentrations of Zr precursors here probably compensate the lower temperature due to the less acidic pHs of the solutions. Indeed, higher pH most likely accelerate the nucleation/growth process as the ligands are more easily deprotonated and the formation of Zr oxo-clusters is favored in such conditions. [28,31] To verify such assumptions, we followed in-situ the nucleation-growth processes at room temperature using PXRD (shown in Figure S17-21) in order to estimate kinetics parameters. It can be concluded that 1) no intermediate states were detected during the synthesis process, 2) nucleation-growth processes of MOF-801 at different concentrations (0.3 mmol/mL and 0.075 mmol/mL) (Figure 4(ab)) were twice longer (48h, 42h, respectively) than that of MOF-801 at 0.15 mmol/mL. This means that further tuning the concentration, higher or lower, is here not advantageous if one wants to faster the synthesis of MOF-801. When considering a higher concentration (0.3 mmol/mL), the solution becomes more acidic (initial pH=0.08), which may slightly inhibit the MOF growth and slow down its crystallization. To confirm this point, a synthesis was performed with the initial reactant concentrations (0.15 mmol/mL) in presence of 0.6 mmol/mL of HCl. The addition of HCl lowers the initial pH to a value similar to the synthesis performed with 0.3 mmol/mL reactant concentrations. The kinetics showed a similar trend in both cases, strongly evidencing the detrimental effect of low inital pH on the reaction kinetics. When using a lower concentration (0.075 mmol/ mL, pH= 0.50), we hypothesize than even if the pH is slightly higher, the high dilution of the reactants may lead to a deceleration of the kinetics. Additionally, when replacing ZrCl4 by the less acidic ZrOCl2 while keeping other parameters unchanged, faster nucleation-growth processes (from 19 h to 9h) were observed as a consequence of the presence of pre-formed Zr-oxo or Zr-OH bonds in ZrOCl2. As a whole, our findings reveal that in addition to the typical parameters investigated so far (temperature, pH, metal source…), the concentration of the reactants is also playing a key role in the synthesis of Zr-MOF, particularly at room temperature. Room temperature synthesis of Metal (IV) based MOFs, with its lower energy penalty and safer conditions, endows a great potential for a more sustainable industrial production, particularly for more robust Zr-MOFs. However, the previous examples of lab-scale synthesis of MOFs at room temperature led in most cases to very low space-time yields (STY), less than 1 kg/m 3 /day, [32] which does not meet at all the needs of industry. In the case of Zr-MOFs, this is mainly due to their prolonged synthesis time at room temperature. [33] Taking benefit from our preliminary crystallization study, we therefore scaled-up the benchmark Zr MOF MOF-801 by using a 5L pilot scale reator with mechanical stirring (Figure 5(a)). The space-time yield of the product was calculated according to the dry solid collected after washing and drying steps, reaching 168 kg/m 3 /day. To the best of our knowledge, this is the first example of room temperature synthesized Zr-MOF with such a high space-time yield. This quality of a scaled-up material in comparison with a small scale laboratory sample is also a prerequisite for industrial applications. As MOF-801 is a prominent example of sorbent suitable for heat reallocation applications, [34] we investigated the water sorption properties (Figure 5(b)) of our scaled up material. Noteworthy it led to a consistently highly crystalline solid in comparison to the labscale synthesis (see Figure S22), attaining a remarkably high water adsorption capacity of 41 wt% H2O/ MOF. The isotherm is of a Type-I shape mainly due to the presence of defects in the structure, which on the one hand increases the hydrophilicity due to the increased Coulomb interaction from the hydroxyl terminal group at the defect sites, while on the other hand expands the pore volume of the MOF due to the missing linker effect aforementioned.

Conclusion

In summary, we report here a new facile and versatile approach for the one-step synthesis of a series of highly porous metal(IV) carboxylate MOFs (M=Zr, Hf, Ce) including five 12-connected and two 8-connected MOFs, using greener ambient temperature conditions. Not only this allowed us to produce high quality crystalline and porous robust metal(IV) carboxylate MOFs with varying metal salts, but also, through a tuning of the synthetic parameters, enabled a control the MOF particle size at the nanoscale. Finally, we evaluated the possibility of scaling-up the synthesis with the use of 5L pilot scale system, evidencing a highest space-time yield for the room temperature synthesis of MOF-801(Zr). As a whole, our method paves the way for the versatile RT synthesis of series of metal(IV)-MOFs that is of strong interest for many applications as well as for their industrial production under sustainable conditions.

Several micro/ mesoporous metal(IV)-based MOFs with different functional groups were synthesized at room temperature using environmentally friendly conditions. The versatility of this strategy allows the use of different reactants and the control of the MOF particle size. A synthesis using a pilot scale-up system demonstrates the potential of this approach for the industrial production of metal (IV)-based MOFs.

Figure 1 .

 1 Figure 1. Schematic diagram of the room temperature one-step approach (a). Representative of the crystal structures of MOF-801(Zr, Hf, Ce) viewed along (101) plane and the illustration of existing defects (b), UiO-66-COOH/ NH2 (c), DUT-67(PDA)(d) andPCN-222 (e). Metal polyhedra, carbon and oxygen atoms are in cyan, black and red, respectively (hydrogen atoms have been omitted for the sake of clarity).

Figure 2 .

 2 Figure 2. PXRD patterns (λCu=1.5406Å) of: (a) MOF-801(Zr), MOF-801(Hf), MOF-801(Ce), (b) UiO-66-NH2 and UiO-66-COOH, (c) DUT-67(PDA) and the corresponding simulated PXRD patterns. N2 adsorption-desorption isotherms (adsorption, filled symbols; desorption, empty symbols) at 77K (P0=1 atm) of: (d) MOF-801(Zr), MOF-801(Hf), MOF-801(Ce), (e) UiO-66-NH2 and UiO-66-COOH, (f) DUT-67(PDA).

Figure 4 .

 4 Figure 4. (a) Plot of synthesis time versus the intensities of the strongest (1, 1, 1) reflection of MOF-801 with varying precursors concentrations (the reactions

Figure 3 .

 3 Figure 3. (a) Experimental and simulated PXRD patterns (λCu=1.5406Å) of PCN-222, (b) N2 adsorption-desorption isotherm at 77K of PCN-222 (P0=1 atm).

Figure 5 .

 5 Figure 5. (a) 5L glass reactor laboratory pilot scale system for the upscaling synthesis of MOF-801-LS (large scale), inserted picture: mass of product after synthesis, washing and drying, (b) Water sorption isotherms (adsorption, filled symbols; desorption, open symbols) of MOF-801-LS (large-scale) and MOF-801 (lab-scale) at 25°C.

Table 1 .

 1 Summary of the properties of room temperature synthesized MOFs presented in this work

Acknowledgements

S. Dai acknowledges the support of CSC scholarship (201706140196).