Moussa Kenawi

Emmanuel Rouger

Marie-Laure Island

Patricia Leroyer

François Robin

Séverine Remy

Laurent Tesson

Ignacio Anegon

Kévin Nay

Frédéric Derbré

Pierre Brissot

Martine Ropert

Thibault Cavey

Olivier Loréal
email: olivier.loreal@univ-rennes1.fr

Affiliation

Ceruloplasmin deficiency does not induce macrophagic iron overload : lessons from a new rat model of hereditary aceruloplasminemia

Keywords: Iron overload, ceruloplasmin, hepcidin, CRISPR/Cas9, genetic disease

Hereditary aceruloplasminemia (HA) related to mutations in the ceruloplasmin gene leads to iron accumulation. Ceruloplasmin ferroxidase activity being considered essential for macrophage iron release, macrophage iron overload is expected, but is not found in hepatic and splenic macrophages in Humans. Our objective was to get a better understanding of the mechanisms leading to iron excess in HA.

A CRISPR/Cas9 knock-out of the Cp gene was performed on Sprague-Dawley rats. We evaluated the iron status in plasma, the expression of iron metabolism genes, and the status of other metals whose interactions with iron are increasingly recognized.

In Cp -/-rats, plasma ceruloplasmin and ferroxidase activity were absent, together with decreased iron concentration and transferrin saturation. Similarly to Humans, the hepatocytes were iron overloaded conversely to hepatic and splenic macrophages. Despite a relative hepcidin deficiency in Cp -/-rats and the loss of ferroxidase activity, potentially expected to limit the interaction of iron with transferrin, no increase of plasma non-transferrin bound iron level was found. Copper was decreased in the spleen while manganese was increased in the plasma. These data suggest that the reported role of ceruloplasmin cannot fully explain the iron hepatosplenic phenotype in HA and encourage to search for additional mechanisms.

Introduction

HA (OMIM # 604290) is a rare genetic disease affecting about one person per two millions (1). The disease is related to homozygous or compound heterozygous mutations within the ceruloplasmin gene (CP) located on chromosome 3 [START_REF] Yang | Characterization, mapping, and expression of the human ceruloplasmin gene[END_REF]. HA is characterized by progressive iron overload targeting especially the liver and brain, and resulting in severe complications at variable ages, including diabetes and neurological disturbances [START_REF] Bosio | Anemia and iron overload due to compound heterozygosity for novel ceruloplasmin mutations[END_REF][START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF]. Despite iron overload, serum iron and transferrin saturation are low and anemia is also observed [START_REF] Bosio | Anemia and iron overload due to compound heterozygosity for novel ceruloplasmin mutations[END_REF][START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF].

The ceruloplasmin gene encodes the ceruloplasmin protein in two different forms resulting from an alternative splicing [START_REF] Patel | Alternative RNA splicing generates a glycosylphosphatidylinositol-anchored form of ceruloplasmin in mammalian brain[END_REF]. The first gene product is mainly expressed by hepatocytes and secreted in plasma [START_REF] Sato | Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin[END_REF]. The second one is characterized by the presence of a glycosylphosphatidylinositol sequence allowing its anchorage in plasmic membrane, especially in astrocytes according to in vitro studies [START_REF] Patel | A novel glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed by mammalian astrocytes[END_REF]. Ceruloplasmin is a multicopper-oxidase that catalyzes oxidation of ferrous iron into ferric iron [START_REF] Osaki | The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum[END_REF]. Ferroxidase activity requires the association of the peptidic sequence with six atoms of copper [START_REF] Sato | Mechanisms of copper incorporation during the biosynthesis of human ceruloplasmin[END_REF][START_REF] Hellman | Mechanisms of copper incorporation into human ceruloplasmin[END_REF]. This biological activity is reported to play a key role in the iron release from cells, especially macrophages, providing iron to plasma for maintaining sufficient amounts of bio-available iron to the cells [START_REF] Harris | Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux[END_REF][START_REF] Young | Ceruloplasmin, transferrin and apotransferrin facilitate iron release from human liver cells[END_REF]. It has been reported that, following the iron release by the iron exporter ferroportin, ceruloplasmin permits, by oxidizing iron, its association to transferrin [START_REF] Osaki | The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum[END_REF]. Each transferrin molecule may link up to two iron atoms, and subsequently delivers iron to every cell in the body [START_REF] Osaki | The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum[END_REF]. Moreover, a role of GPI-ceruloplasmin in the stabilization of ferroportin on cell membrane has also been reported [START_REF] De Domenico | Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin[END_REF][START_REF] Kono | Biological effects of mutant ceruloplasmin on hepcidin-mediated internalization of ferroportin[END_REF]. Such a biological role suggests that, in HA, iron should be retained within macrophagic cells similarly to the reported findings in the ferroportin disease in which the activity of the iron exporter is strongly decreased [START_REF] Pietrangelo | The ferroportin disease. Blood Cells[END_REF][START_REF] Pietrangelo | Ferroportin disease: pathogenesis, diagnosis and treatment[END_REF]. Similarly, during inflammatory process characterized by high hepcidin levels, iron retention is observed within macrophages due to hepcidin increase [START_REF] Nemeth | IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin[END_REF][START_REF] Wrighting | Interleukin-6 induces hepcidin expression through STAT3[END_REF] that induces internalization and degradation of ferroportin [START_REF] De Domenico | The Molecular Mechanism of Hepcidin-mediated Ferroportin Down-Regulation[END_REF][START_REF] Theurl | Autocrine formation of hepcidin induces iron retention in human monocytes[END_REF]. However, from published HA case reports, it appears that, in the liver, the Kupffer cells are almost constantly spared by iron excess [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF][START_REF] Skidmore | Aceruloplasminaemia with progressive atrophy without brain iron overload: treatment with oral chelation[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF][START_REF] Piperno | Aceruloplasminemia: waiting for an efficient therapy[END_REF], and that, in the spleen, there is no increase of iron concentration [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF][START_REF] Skidmore | Aceruloplasminaemia with progressive atrophy without brain iron overload: treatment with oral chelation[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF], contrasting with the observations in the ferroportin disease [START_REF] Pietrangelo | The ferroportin disease. Blood Cells[END_REF][START_REF] Pietrangelo | Ferroportin disease: pathogenesis, diagnosis and treatment[END_REF].

Therefore, the hepatosplenic phenotype of iron overload observed in human HA does not fit with the reported biological role of ceruloplasmin, and our aim was to investigate the mechanisms contributing to iron misdistribution between spleen and liver, in order to progress in the understanding of the biological role of ceruloplasmin and of the mechanisms underlying the development of HA. It is noteworthy that ceruloplasmin knock-out mouse models give contrasted data regarding the iron overload phenotype in liver and spleen. Indeed, some animal data shows only hepatic iron overload, with no significant iron concentration increase in the spleen [START_REF] Jiang | Hephaestin and ceruloplasmin play distinct but interrelated roles in iron homeostasis in mouse brain[END_REF][START_REF] Meyer | Copper transport and metabolism are normal in aceruloplasminemic mice[END_REF][START_REF] Gouya | Genetic study of variation in normal mouse iron homeostasis reveals ceruloplasmin as an HFE-hemochromatosis modifier gene[END_REF]. Other reports describe both hepatic and splenic iron overload A c c e p t e d M a n u s c r i p t [START_REF] Harris | Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux[END_REF][START_REF] Chen | Age-related changes in iron homeostasis in mouse ferroxidase mutants[END_REF], with, in the liver, presence of iron within hepatic macrophages [START_REF] Harris | Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux[END_REF][START_REF] Yamamoto | Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice[END_REF]. Thus, these data contrasts with the clinical findings in humans [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF][START_REF] Skidmore | Aceruloplasminaemia with progressive atrophy without brain iron overload: treatment with oral chelation[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF], and suggests that mice could be not fully appropriate to mimic human HA. We hypothesized that a rat model could provide relevant informations to understand the disease ; thus, we used the rat as an alternative model to characterize the disease. We selected the Sprague-Dawley rat in which the type of hepatic iron load phenotype obtained during oral iron supplementation is very similar to the human profile both during genetic hemochromatosis and in excessive oral iron intake [START_REF] Bacon | Hepatic lipid peroxidation in vivo in rats with chronic iron overload[END_REF]. Therefore, the CRISPR/Cas9 technology was used to generate and study a new rat aceruloplasminemic model aiming to mimic the unexpected human aceruloplasminemic hepato-splenic iron phenotype and to characterize the impact of aceruloplasminemia on other metals involved in biological processes (Cu, Zn, Mn, Mo, Co) that could play a role during aceruloplasminemia.

A c c e p t e d M a n u s c r i p t

Material and Methods

Development of an aceruloplasminemic animal model

Sprague-Dawley (SD/Crl) rats were obtained from Charles River (L'Arbresle, France). All the animal care and procedures were approved by the Animal Experimentation Ethics Committee of the Pays de la Loire region, France, in accordance with the guidelines from the French National Research Council for the Care and Use of Laboratory Animals (Permit Number: CEEA-PdL-2015-692). The sgRNA was designed and produced by the facility TACGENE. The sgRNAcr377 used targeted the exon 1 of ceruloplasmin gene and had the following sequence : GGAATTACTGAAGCAGTTT. The sgRNA (200 ng/µl) and Cas9 protein (3µM) were incubated at room temperature for 10 min to allow formation of ribonucleoprotein complexes and then were kept at 4 °C until microinjection. Process and microinjection were performed as reported previously [START_REF] Remy | Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation[END_REF]. Briefly, zygotes were collected from pre-pubescent (4-5 weeks old) SD donor female rats, super-ovulated by IP injection of pregnant mare serum gonadotropin (30 IU; Intervet, France) and 48h later, of human chorionic gonadotropin (20 IU; Intervet, France) that were mated with SD fertile males. One-cellstage fertilized embryos were sequentially microinjected into the male pronucleus and into the cytoplasm.

The same day, surviving embryos were implanted within pseudo-pregnant females (0.5 dpc) oviduct of and growed until full-term.

Genotyping

Tail biopsies from 8-to 10-day-old rats were digested overnight at 56 °C, in 500 µL of tissue digestion buffer (0.1M Tris-HCl pH 8.3, 5 mM EDTA, 0.2% SDS, 0.2M NaCl, 100 µg/ml proteinase K).

The CRISPRs nuclease targeted regions were PCR amplified from diluted lysed samples (1/20) with a highfidelity polymerase (Herculase II fusion polymerase). To detect gene edition, locus specific primers were used and mutations were analyzed by HMA-CE and direct sequencing of PCR products.

Characterization of aceruloplasminemic rats

From this founder, after crossing with wild-type SD rats, three groups (wild-type, heterozygotes, and homozygotes) of 10 rats each were obtained. The genotype of animals was performed by PCR using primers. DNA extractions were performed from young rat tails by using the Nucleospin Tissue® kit (Macherey Nagel), then dosed with Spectrometer ND-1000® (Nanodrop). PCR were realized by using 200 ng of DNA and by using specific primers: forward primer (GGGCTCCAAGAGGAAGAAAC), reverse primer (ATTTGGCCCATAAACAACAAAGAA), and GoTaq G2 Hot Start Green Mastermix® (Promega) on a Applied Biosystem Veriti 96 well Thermal Cycler® (ThermoFisher) with the following sequence : 3 minutes of heating at 95°C, followed by 30 cycles composed of 1 minute at 95°C, 1 minute at 62°C, and 2 minutes at 72°C, before a final single step at 72°C for 5 minutes. PCR products were investigated on 3% A c c e p t e d M a n u s c r i p t agarose gel. Gels were exposed to U.V. in a transilluminator (Fisher scientific), and blots pictures were analyzed with Vision-Capt® software.

All studied animals were 6 month-old males. During breeding, animals were submitted at reversed lighting, had free access to water and standard rat food, and had benefited of litter changing one time a week. A standard diet, designed to support growth and maintenance (Teklad Global 16% protein Rodent Diet, Envigo, USA) contained 200 mg/kg of iron, 15 mg/kg of copper, 100 mg/kg of manganese, and 70 mg/kg of zinc. The protocol was validated by the Animal Experimentation Ethics Committee of Région Bretagne, France (referral number : 8554).

Animals were fasted 10 hours and anesthetized by isoflurane. After a laparotomy from symphysis to xiphoid appendix 1 ml of portal blood was collected. Then, cardiac blood sampling was performed in two successive phases : 1 ml on non-heparinized syringe and a larger volume on sodium-heparinate syringe respectively leading to cardiac arrest and animal death. The different organs were then collected, each one was rapidly split in two parts, and placed either in formaldehyde 4% or in liquid nitrogen.

Sample conditioning and storage

For serum preparation, after 2 hours at room temperature, non-heparinized blood samples were centrifuged (200 G; 10 minutes). Supernatants were collected, (centrifuged at 800 G ; 3 minutes), then aliquoted, and finally stored at -80°C until use.

For plasma preparation and erythrocytes cleaning, heparinized blood samples were centrifuged 10 minutes at 2230 G, (20°C). Supernatants were then collected and aliquoted in 100 µl to 600 µl samples, before storage at -80°C. Three to four ml of NaCl 0,9% were added for washing of blood cells pellets, then gently mixed to these last ones, and centrifuged at 1833 G during 10 minutes at 20°C. The washing was repeated three times. Then blood cells pellets were collected and aliquoted and immediately stored at -80°C. Tissues collected in liquid nitrogen were stored at -80 °C.

Haemoglobin measurement

Haemoglobin concentration was measured in the clinical analysis platform of Rennes University Hospital, by a spectrophotometry method using an ABL 90® robot (Radiometer).

Biochemical assays

Plasma measurements were performed in the clinical analysis platform of Rennes University Hospital. Ceruloplasmin assays were performed by immunoturbidimetric method by a Cobas® 8000 robot (Roche), using a Ceruloplasmin 100 tests® kit (Roche) that contained a rabbit anti-human ceruloplasmin antibody. Serum and plasma iron concentrations, as well as unsaturated iron bound capacity (UIBC) values, were obtained by colorimetric method using respectively the kits Iron Gen.2, 750 tests® (Roche) and

A c c e p t e d M a n u s c r i p t

Unsaturated Iron-Binding Capacity 100 tests® (Roche), both performed by a Cobas® 8000 robot (Roche) according to the principles previously reported [START_REF] Cavey | Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships[END_REF]. Transferrin saturation was calculated from UIBC value by the following equation : [iron/(iron+UIBC)]x100. Ferroxidase activity was measured on plasma samples by spectrophotometry using a Cobas® C111 robot (Roche), according to the method previously reported [START_REF] Lainé | Serum ceruloplasmin and ferroxidase activity are decreased in HFE C282Y homozygote male iron-overloaded patients[END_REF]. Non-transferrin bound iron (NTBI) was quantified in serum rat samples by a previously reported method [START_REF] Breuer | A fluorescence-based one-step assay for serum non-transferrinbound iron[END_REF].

Quantification of metals in tissues and erythrocytes

For each tissue sample, a biopsy fragment of about 3 x 3 mm was cut and desiccated at 120°C during 10 to 15 hours. Samples were then recut for obtaining a weight between 0.5 and 3 mg. For erythrocytes, 250 µl of each sample were used. Assays were performed using ICP/MS apparatus equipped with a collision reaction cell as previously reported [START_REF] Cavey | Mouse genetic background impacts both on iron and non-iron metals parameters and on their relationships[END_REF].

Histological analysis

Formaldehyde fixed biopsies were paraffin embedded. Four µm organ slices were performed and there stained by using Hemalun-eosin-safron and Perls methods in the H2P2 platform (UMS Biosit, Rennes).

Real-time qPCR

Total RNAs were extracted following the procedure of the Nucleospin RNA Plus® (Macherey Nagel) extraction kit, and then measured with Spectrometer ND-1000® (Nanodrop).

Reverse transcriptase step was performed by using the Hight Capacity cDNA Reverse Transcriptase® (Applied Biosystem) kit for obtaining 2 µg of cDNA per sample of 20 µl before a final 1/6 dilution in nuclease free water. qPCRs were realized using 384 wells plates, with 2 µl of sample, 1 µl of forward primer then 1 µl of reverse primer, and 5 µl of SYBR GREEN 1 per well. Plates were then covered with a transparent film and centrifuged during 1 minute at 1000 rpm. Runs were performed in a 7900 HT Fast Real-time PCR system® (ThermoFisher) and data processed with SDS 2.4® software. Housekeeping genes used were Hprt1, Tbp, and Gusb ; Delta Ct were calculated by subtracting for each sample the Ct average of these three genes to the Ct of target gene. Primer pairs used are indicated in a Table (Supp. data Table 1).

Western-blotting

Cell membrane protein extraction was performed as previously reported, but at 100 000 g for one hour [START_REF] Canonne-Hergaux | Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron[END_REF]. Total protein extraction and western-blotting using 10 percent acrylamide gels were performed as described previously [START_REF] Salaun | Myriocin prevents muscle ceramide accumulation but not muscle fiber atrophy during shortterm mechanical unloading[END_REF]. The following antibodies were used : Ferroportin (1:500 ; Alomone Labs # Hsc70 (1:5000 ; Santa Cruz # sc-7298). We used this p-ERK1/2-Thr202-Tyr204 antibody [START_REF] Cornejo | Chronic iron overload enhances inducible nitric oxide synthase expression in rat liver[END_REF][START_REF] Chen | Iron causes interactions of TAK1, p21ras, and phosphatidylinositol 3-kinase in caveolae to activate IκB kinase in hepatic macrophages[END_REF][START_REF] Zhang | The action of JAK, SMAD and ERK signal pathways on hepcidin suppression by polysaccharides from Angelica sinensis in rats with iron deficiency anemia[END_REF][START_REF] Poli | Transferrin receptor 2 and HFE regulate furin expression via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) signaling. Implications for transferrindependent hepcidin regulation[END_REF][START_REF] Tangudu | Deregulation of Hepatic Mek1/2-Erk1/2 Signaling Module in Iron Overload Conditions[END_REF][START_REF] Wallace | Combined deletion of Hfe and transferrin receptor 2 in mice leads to marked dysregulation of hepcidin and iron overload[END_REF], as well as this p-STAT3-Tyr705 antibody [START_REF] Cavey | Simulated microgravity decreases circulating iron in rats: role of inflammation-induced hepcidin upregulation[END_REF][START_REF] Armitage | Hepcidin regulation by innate immune and infectious stimuli[END_REF][START_REF] De Domenico | Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice[END_REF][START_REF] Huang | Contribution of STAT3 and SMAD4 pathways to the regulation of hepcidin by opposing stimuli[END_REF], on the basis of their use in rats, as reported in the literature. We tested the p-SMAD1/5/8-Ser463/465 in human HepG2 hepatic cells that were exposed or not to BMP6, an inducer of SMAD1/5/8 phosphorylation [START_REF] Kersten | BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1[END_REF]. By western blot, we clearly found a signal increase at about 55 kDa in the sample treated by BMP6, compared to the untreated sample (Data not shown). We tested the expression of ferroportin by western blot in spleen and liver samples and found a significant expression at the expected molecular weight close to 60 kDa ; the signal totally disappeared when the peptide used for immunization was added during antibody incubation. Regarding hephaestin, we analyzed rat spleen and liver and found, at the expected molecular weight (close to 130 kDa), a significant expression of the protein in the spleen, known to express hephaestin, and not in the liver that express very low levels of hephaestin mRNA [START_REF] Li | Quantitative study of iron metabolism-related genes expression in rat[END_REF][START_REF] Zhang | Localization of iron metabolismrelated mRNAs in rat liver indicate that HFE is expressed predominantly in hepatocytes[END_REF]. The blot quantification analyses were conducted using Image Gauge® software (Fujifilm).

Statistical analysis

All the data were analyzed statistically with SPSS® software, using non-parametric tests. The three groups were submitted to Kruskal-Wallis test and Mann-Whitney test when appropriate. p values <0.05 were considered statistically significant. Data are presented as means ± SD.

A c c e p t e d M a n u s c r i p t

Results

Generation of ceruloplasmin-deficient rats.

A sgRNA targeting exon 1 of the ceruloplasmin (Cp) gene (Fig. 1 A andB) was designed, then microinjected complexed with Cas9 in rat zygotes (n=189) and implanted (n=154) in pseudopregnant females. Offspring (n=47) were genotyped and resulted in the generation of mutated animals (n=30) with different types in insertions and deletions (Data not shown). Among these mutated rats, one of them showed a mutation that resulted in a 54 base pair deletion and a 2 base pair insertion in exon 1 of the ceruloplasmin gene (Fig. 1 C). This resulted in the disruption of the coding frame and the appearance of a premature stop codon occurring in exon 2 (Fig. 1 D) with a predicted degradation of the whole mRNA due to the widely distributed mechanism of nonsense-mediated mRNA decay [START_REF] Frischmeyer | Nonsense-mediated mRNA decay in health and disease[END_REF].

This founder animal was crossed with wild-type animals and the mutation was observed in the F1 offspring. F1 heterozygous mutated animals were crossed to obtain homozygous mutated animals (Fig. 1 E).

Characterisation of the iron phenotype in aceruloplasminemic rat model

Plasma ceruloplasmin assays showed very low ceruloplasmin concentrations in homozygotes, and significantly lowered values in heterozygotes compared to wild-type rats (Fig. 2 A). Ferroxidase activity was almost undetectable in homozygotes, and half preserved in heterozygote animals (Fig. 2 B).

Plasma iron concentration was significantly decreased in homozygotes and fully preserved in heterozygotes compared to wild-types, both in peripheral and portal plasma (Fig. 2 C andD). Transferrin saturation followed the same variations (Fig. 2 E andF). NTBI levels were decreased both in portal and systemic plasma in homozygotes as compared to wild-types and heterozygotes (Fig. 2 G andH).

Hemoglobin concentrations were not affected in homozygous and heterozygous groups as compared to controls, and erythrocyte iron concentration was not modified by the presence of the mutation at heterozygous or homozygous state (Fig. 2 I andJ).

Liver iron concentration (LIC) was significantly higher in homozygous rats, whereas spleen iron concentration (SIC) was significantly lower compared to values found in wild-type animals ; in heterozygous rats, both LIC and SIC were not affected compared to wild-type animals (Fig. 2 K andL).

Perls staining showed liver iron deposits in homozygotes, located in hepatocytes and sparing Kupffer cells (Fig. 3 A andB). Spleen iron deposits, mainly located in macrophages from the red pulp, appeared to be lower in homozygotes than in wild-type animals (Fig. 3 C andD).

Characterization of the non-iron metal phenotype in aceruloplasminemic rats

Plasma copper concentrations were strongly decreased in homozygotes, and half preserved in heterozygotes, while erythrocyte copper concentrations were not affected in mutated animals (Fig. 4 A andA c c e p t e d M a n u s c r i p t B). Liver copper concentration was unchanged, while spleen copper was significantly lower in homozygotes and heterozygotes compared to wild-type animals (Fig. 4 C andD).

Concentrations of other metals including molybdenum, manganese, zinc and cobalt were also investigated in liver, spleen, erythrocytes and plasma of animals (Supp. data Table 2). In homozygous animals, a significant increase of cobalt and manganese concentrations was found in erythrocytes and plasma, as compared to heterozygous and controls (Fig. 4 E andF).

Impact of aceruloplasminemia on the expression of iron metabolism genes

In the liver, hepcidin antimicrobial peptide (Hamp) mRNA levels were slightly but significantly decreased in homozygous as compared to heterozygous animals, but not versus wild-type rats (Fig. 5 A). The ratio between Hamp mRNA and LIC levels was strongly decreased in homozygous animals as compared to wild-type and heterozygous animals (Fig. 5 B). Hepatic mRNA levels of transferrin receptor 2 (Tfr2), hemojuvelin BMP co-receptor (Hjv), and Hfe, (Supp. data Figure 1) did not change between the groups. Levels of phosphorylated forms of ERK1/2, STAT3, and SMAD1/5/8, proteins did not show significant variations between wild-type and homozygous animals (Fig. 5 C ; Supp. data Figure 2). Transferrin receptor (Tfrc), solute carrier family 11 member 2 (Slc11a2), solute carrier family 39 member 14 (Slc39a14) (Supp.

Data Figure 1), solute carrier family 40 member 1 (Slc40a1) and hephaestin (Heph) (Fig. 5 D andE) mRNA levels, were not affected in homozygous and heterozygous animals. It is noteworthy that hepatic Heph mRNA levels were particularly low in all groups. The hepatic expression of cell membrane ferroportin was not significantly modulated in aceruloplasminemic rats (Fig. 5 F ; Supp. data Figure 2). Hephaestin cell membrane protein was undetectable both in wild-type and homozygous groups in the liver (data not shown).

In the spleen, the mRNA levels of Tfrc, Slc11a2 (Supp. Data Figure 1), and Heph (Fig. 5 H), were not different between the three groups. mRNA levels of Slc40a1, were slightly but significantly increased in homozygous as compared to wild-type animals, only (Fig. 5 G). Ferroportin and hephaestin cell membrane protein expression were not significantly modulated in homozygous animals (Fig. 5 I ; Supp. data Figure 2).

In the duodenum, mRNA levels of Slc11a2 (Supp. data Figure 1), Slc40a1 and Heph (Fig. 5

J and K)

were not affected by homozygous or heterozygous state. Ferroportin and hephaestin cell membrane protein expression were unchanged between wild-type and homozygous animals (Fig. 5 L ; Supp. data Figure 2).

Impact of aceruloplasminemia on the expression of copper metabolism genes

Hepatic mRNA levels of the two genes encoding factor V and factor VIII that are multicopper oxidases were unchanged in homozygous and heterozygous animals, compared to controls (Supp. data Figure 3). mRNA levels of Atp7a, Atp7b, and Slc31a1 genes that encode copper transporters were unchanged in both liver and spleen (Supp. data Figure 3).

A c c e p t e d M a n u s c r i p t

Discussion

Our data, obtained in a new aceruloplasminemic rat model, shows that serum biochemical parameters obtained in the Cp -/-were in accordance with those reported in humans [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF] and in one mouse model [START_REF] Guo | Hepcidin, an antimicrobial peptide is downregulated in ceruloplasmin-deficient mice[END_REF]. Indeed, both ceruloplasmin and ferroxidase activity were undetectable. Moreover, plasma iron concentration and transferrin saturation levels were significantly decreased in homozygous animals only, supporting a strong decrease of plasma iron bioavailability. Importantly, there were neither anemia nor reduction of erythrocyte iron content in our model, in contrast with human findings [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF][START_REF] Ogimoto | Criteria for early identification of aceruloplasminemia[END_REF] and some mouse models [START_REF] Yamamoto | Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice[END_REF][START_REF] Cherukuri | Anemia and impaired stress-induced erythropoiesis in aceruloplasminemic mice[END_REF][START_REF] Fuqua | Severe iron metabolism defects in mice with double knockout of the multicopper ferroxidases hephaestin and ceruloplasmin[END_REF], but in accordance with another mouse model [START_REF] Harris | Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux[END_REF].

Our findings with, in the liver, a highly predominant hepatocyte iron overload, and, in the spleen, a decrease of iron concentration, fully fit the human disease [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF][START_REF] Skidmore | Aceruloplasminaemia with progressive atrophy without brain iron overload: treatment with oral chelation[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF], whereas data obtained in mice are very contrasted [START_REF] Harris | Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux[END_REF][START_REF] Jiang | Hephaestin and ceruloplasmin play distinct but interrelated roles in iron homeostasis in mouse brain[END_REF][START_REF] Meyer | Copper transport and metabolism are normal in aceruloplasminemic mice[END_REF][START_REF] Gouya | Genetic study of variation in normal mouse iron homeostasis reveals ceruloplasmin as an HFE-hemochromatosis modifier gene[END_REF][START_REF] Chen | Age-related changes in iron homeostasis in mouse ferroxidase mutants[END_REF][START_REF] Yamamoto | Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice[END_REF]. We must underline that in mice, the genetic background in inbred strains plays a major role as a modifier of the iron phenotype [START_REF] Dupic | Inactivation of the hemochromatosis gene differentially regulates duodenal expression of iron-related mRNAs between mouse strains[END_REF][START_REF] Dupic | Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice[END_REF]. Conversely, our data have been obtained in an outbred strain that keeps a significant genetic background variability that may help to identify strong elements involved in the development of the phenotype [START_REF] Brower | Comparative analysis of growth characteristics of Sprague Dawley rats obtained from different sources[END_REF]. Indeed, it has been recently reported that for most applications in animal studies, the use of robust and diverse subjects is preferred to obtain generalizable conclusions across conditions and populations [START_REF] Tuttle | Comparing phenotypic variation between inbred and outbred mice[END_REF]. It has been proposed that, contrary to classical expectations, the adoption of outbred animals as research subjects may improve experimental replicability [START_REF] Tuttle | Comparing phenotypic variation between inbred and outbred mice[END_REF]. Moreover, it should be outlined that, conversely to rats and Humans, in mice, iron deposits during iron overload may be localized in the nucleus of hepatocytes, suggesting significant differences in the iron metabolism of mouse hepatocytes [START_REF] Smith | Characterization and accumulation of ferritin in hepatocyte nuclei of mice with iron overload[END_REF]. In addition, there is only one gene of hepcidin in rats like in Humans, in contrast with mice that exhibit two genes, the role of the second one being not yet clearly established [START_REF] Ilyin | Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload[END_REF][START_REF] Courselaud | Strain and gender modulate hepatic hepcidin 1 and 2 mRNA expression in mice[END_REF]. Therefore, we thought that a rat model would be valuable to get complementary and relevant informations for a better understanding of ceruloplasmin functions involved in HA.

Our histological data, as reported in Humans [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF][START_REF] Skidmore | Aceruloplasminaemia with progressive atrophy without brain iron overload: treatment with oral chelation[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF], showing that iron deposits are mainly located in hepatocytes whereas both Kupffer cells, the resident macrophages in the liver, and spleen macrophages did not exhibit significant iron deposits compared to controls, contrast with the classical pathophysiological hypothesis postulating iron retention in macrophages during HA [START_REF] De Domenico | Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin[END_REF]. Our data strongly suggests that, during HA, iron is not retained within macrophage despite the strong decrease of ferroxidase activity. These findings contrast with those in mice showing that iron egress is decreased in macrophages when ferroxidase activity is altered [START_REF] Chen | Age-related changes in iron homeostasis in mouse ferroxidase mutants[END_REF]. However, they are in agreement with other mouse reports [START_REF] Jiang | Hephaestin and ceruloplasmin play distinct but interrelated roles in iron homeostasis in mouse brain[END_REF][START_REF] Meyer | Copper transport and metabolism are normal in aceruloplasminemic mice[END_REF][START_REF] Gouya | Genetic study of variation in normal mouse iron homeostasis reveals ceruloplasmin as an HFE-hemochromatosis modifier gene[END_REF], suggesting that iron egress is not altered in macrophages.

A c c e p t e d M a n u s c r i p t

Therefore, complementary mechanisms contributing to the hepatocyte iron load must be searched.

During genetic hemochromatosis, the archetype of hepatocyte iron overload, the following mechanistic sequence is involved in the iron loading process : hepcidin deficiency, related to mutations in the Hfe gene, but also in Hjv, Hamp or Tfr2 genes, leads to an increase of transferrin saturation with appearance of NTBI [START_REF] Brissot | Non-transferrin bound iron: A key role in iron overload and iron toxicity[END_REF], quickly taken up by hepatocytes through the ZIP 14 protein [START_REF] Jenkitkasemwong | SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis[END_REF].

Our data shows that the homozygous Cp -/-animals presented a relative hepcidin deficiency, as indicated by a drop of the hepcidin on LIC ratio, reflecting the absence of the expected increase of hepcidin mRNA levels in the liver of iron overloaded animals. Moreover, we did not find any modulation of Hfe, Hjv and Tfr2 hepatic mRNA steady-state levels that could have participated to the genesis of the relative hepcidin deficient state. In addition, the transduction pathways that depend on these genes did not seem affected as suggested by the absence of phosphorylation level decrease of ERK1/2, SMAD1/5/8 and STAT3.

Whether an inhibitory signal resulting from an erythropoietic effect of the disease is involved, even in absence of anemia, deserves further investigation. It is noteworthy that, whereas the expression of the iron exporter ferroportin, (the hepcidin target) on cell membrane has been reported in cell models to be impaired in macrophages in case of ceruloplasmin deficiency [START_REF] De Domenico | Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin[END_REF][START_REF] Kono | Biological effects of mutant ceruloplasmin on hepcidin-mediated internalization of ferroportin[END_REF][START_REF] Marques | Immune cells and hepatocytes express glycosylphosphatidylinositol-anchored ceruloplasmin at their cell surface[END_REF], we did not find any modulation of ferroportin mRNA and protein levels in the spleen, that is particularly rich in macrophages, and in the liver.

Ferroxidase activity being reported as required for the iron association to transferrin [START_REF] Brissot | 2) Iron metabolism and related genetic diseases: A cleared land, keeping mysteries[END_REF], we looked for an increase of NTBI in the serum of homozygous animals, despite the absence of transferrin saturation increase. Similarly to what has been reported in Humans (4), we did not find any NTBI increase not only in systemic blood flow, but also in portal flow that receives iron from spleen and digestive tract. Moreover, the mRNA levels of Slc39a14, the specific transporter of NTBI [START_REF] Jenkitkasemwong | SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis[END_REF], was not modulated in the liver of Cp -/- animals. Taken together, these data suggests that (an) alternative mechanism(s) allow(s) the transfer of iron on transferrin. In this context, it is noteworthy that the expression level of hephaestin that could be involved in a compensation of ceruloplasmin deficiency was not significantly increased in the spleen and in the duodenum in aceruloplasminemic rats. Moreover, the ferroportin expression level was not affected in spleen, liver and duodenum.

Finally, considering strong relationships between ceruloplasmin and Cu and previous reports underlying potential interactions between the metabolism of iron and those of other metals (65), we investigated the concentration of these metals in plasma and tissues in order to identify elements that could play a role in the development of the disease. We therefore explored whether aceruloplasminemic condition modulated the concentrations of Cu, Mn, Zn Co, Mo in liver, spleen, erythrocytes, and plasma.

The multicopper-oxidase, ceruloplasmin is the most important carrier of Cu in plasma, described as containing about 93 percent of this metal [START_REF] Cartwright | Copper metabolism in normal subjects[END_REF], in accordance with lowered plasma copper concentrations in HA patients [START_REF] Loréal | Aceruloplasminemia: new clinical, pathophysiological and therapeutic insights[END_REF][START_REF] Finkenstedt | Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation[END_REF][START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF][START_REF] Ogimoto | Criteria for early identification of aceruloplasminemia[END_REF], and with animal data [START_REF] Meyer | Copper transport and metabolism are normal in aceruloplasminemic mice[END_REF]. Therefore, as expected, we found a strong decrease of A c c e p t e d M a n u s c r i p t plasma Cu in aceruloplasminemic animals. Regarding the liver, HA case reports are not univocal on copper concentrations, that have been found stable [START_REF] Kono | Hepatic iron overload associated with a decreased serum ceruloplasmin level in a novel clinical type of aceruloplasminemia[END_REF] or increased (3), As previously described in HA mice models [START_REF] Meyer | Copper transport and metabolism are normal in aceruloplasminemic mice[END_REF][START_REF] Yamamoto | Quantitative evaluation of expression of iron-metabolism genes in ceruloplasmin-deficient mice[END_REF], liver copper concentrations in our Cp -/-rats did not show any significant variations. However, we found a decrease of spleen copper concentrations in Cp -/-and Cp +/-animals compared to wildtype rats, contrasting with a previous report in HA mice [START_REF] Meyer | Copper transport and metabolism are normal in aceruloplasminemic mice[END_REF]. It should be noted that this variation affected both Cp -/-and Cp +/-animals, whereas, when analyzing plasma and tissue iron parameters, significant variations were only found in Cp -/-animals. Therefore, it can be hypothezised that the decrease of ceruloplasmin is involved in the spleen copper decrease. This decrease in spleen copper content does not seem related to a lowered copper entry into spleen macrophages during the erythrophagocytosis process, as suggested by the equal erythrocyte copper contents between the three groups.

It is noteworthy that in Cp -/-animals, the plasma manganese levels were found to be increased. The role of ferroportin in Mn egress being supported by previous data [START_REF] Loréal | Iron, hepcidin, and the metal connection[END_REF], this observation supports the absence of direct effect of ceruloplasmin on metal egress. Interestingly, we did not find an overexpression of hephaestin that could have compensated the absence of ceruloplasmin in the spleen, both at mRNA and protein levels.

Whether these alterations play a role during HA will require further investigations.

In conclusion, this new aceruloplasminemic rat model mimics the human hepatosplenic iron load phenotype during HA. Our data suggests that iron overload in this disease is not primarily related to iron retention within macrophages. Whether hepcidin deficiency plays, despite the absence of NTBI, a role in the development of hepatocyte iron load remains to be clarified. Elucidation of the mechanisms involved in these iron metabolism disturbances could help to further understand how iron develops in the brain of human aceruloplasminemia, causing devastating complications. Liver Slc40a1 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (D). Liver Heph mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (E). Representative example of liver ferroportin blots with Hsc70 as load control protein (F). Spleen Slc40a1 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (G). Spleen Heph mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (H). Representative example of spleen ferroportin and hephaestin blots with Hsc70 as load control protein (I). Duodenum Slc40a1 mRNA levels expressed in negative delta cycle threshold, n=10 in CP+/+ and CP+/-groups, n=9 in CP-/-group (J). Duodenum Heph mRNA levels expressed in negative delta cycle threshold, n=10 in CP+/+ and CP+/-groups, n=9 in CP-/-group (K).

A c c e p t e d M a n u s c r i p t

Representative example of duodenum ferroportin and hephaestin blots with Hsc70 as load control protein Liver Hjv mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (B). Liver Hfe mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (C). Liver Tfrc mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (D). Liver Slc11a2 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (E). Liver Slc39a14 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (F). Spleen Tfrc mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (G). Spleen Slc11a2 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (H). Duodenum Slc11a2 mRNA levels expressed in negative delta cycle threshold, n=10 in CP +/+ and CP +/-groups, n=9 in CP -/-group (I).

G H

Supplemental data Figure 3 : Liver F5 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (A). Liver F8 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (B). Liver Atp7a mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (C). Spleen Atp7a mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (D). Liver Atp7b mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (E). Spleen Atp7b mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (F). Liver Slc31a1 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (G). Spleen Slc31a1 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (H).

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

A c c e

  p t e d M a n u s c r i p t AIT001), p-SMAD1/5/8-Ser 463/465 (1:1000 ; Abcam # ab92698), p-STAT3-Tyr 705 (1:1000 ; Cell Signaling # 9131), p-ERK1/2-Thr 202 -Tyr 204 (1:1000 ; Cell Signaling # 4376), Hephaestin (1:250 ; Abcam # ab108003),

Figure 1 :

 1 Figure 1 : A global location of sgRNA in exon 1 is schematically presented (A) The CRISPR/Cas9 process is depicted in the region around the sgRNA targeted site, in exon 1 of ceruloplasmin gene (B). The resulting mutation selected for our model, and the stop codon occurring in exon 2 due to the frame shift, are also represented (C). The frame shift resulting protein is exhibited and compared to the wild-type sequence (D). Example of agarose gel picture representing the three genotypes used in the present study (E). Blots "a" from Cp +/+ rats can be recognized by a band of about 200 bp ; Cp -/animals are characterized by a lower

Figure 2 :Figure 3 :

 23 Figure 2 : Consequences of the mutation on ceruloplasmin protein and activity, plasma iron bio-availability, and tissue iron concentrations. Plasma ceruloplasmin concentration, n=10 in all groups (A). Total ferroxidase activity, n=10 in all groups (B). Peripheral plasma iron concentration, n=10 in all groups (C). Portal plasma iron concentration, n=10 in CP +/+ group, n=9 in CP +/and CP -/groups (D). Peripheral plasma transferrin saturation, n=10 in all groups (E). Portal plasma transferrin saturation, n=10 in CP +/+ group, n=9

Figure 4 :

 4 Figure 4 : Plasma copper concentration, n=10 in all groups (A). Erythrocyte copper concentrations, n=10 in all groups (B). Liver copper concentrations, n=10 in all groups (C). Spleen copper concentrations, n=10 in CP +/+ and CP +/groups, n=8 in CP -/group (D). Erythrocyte cobalt concentrations, n=10 in all groups (E). Plasma manganese concentrations, n=10 in all groups (F). (*, p<0,05 ; **, p<0,01 ; ***, p<0,001) A c c e p t e d M a n u s c r i p t

Figure 5 :

 5 Figure 5 : Liver Hamp mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (A).Ratio between liver Hamp mRNA levels and liver iron concentration, n=10 in all groups (B). Representative examples of liver p-ERK1/2, p-STAT3, and p-SMAD1/5/8 blots with Hsc70 as load control protein (C).

Figure 1 part 1 A

 11 Figure 1 part 1

1 :

 1 Liver Tfr2 mRNA levels expressed in negative delta cycle threshold, n=10 in all groups (A).

2 :

 2 Liver p-ERK1/2 protein levels expressed in fold-change, n=8 in all groups (A). Liver p-STAT3 protein levels expressed in fold-change, n=8 in all groups (B). Liver p-SMAD1/5/8 protein levels expressed in fold-change, n=8 in all groups (C). Liver ferroportin protein levels expressed in fold-change, n=8 in all groups (D). Spleen ferroportin protein levels expressed in fold-change, n=8 in all groups (E). Spleen hephaestin protein levels expressed in fold-change, n=8 in all groups (F). Duodenum ferroportin protein levels expressed in fold-change, n=7 in CP +/+ group, n=6 in CP -/-group (G). Duodenum hephaestin protein levels expressed in fold-change, n=7 in CP +/+ group, n=6 in CP -/-group (H).

Table 1 :

 1 Sequences of forward and reverse primers used for real time qPCR.

	Gene	Forward primer	Reverse primer
	Atp7a	TGGCTGGAACACATAGCGAA	TCCACTTGTTCTTCACTCAGGA
	Atp7b	CAAGGTCATCGAGGAAATCGGC	CATGACGGGGATGCCAAACA
	F8	TGCCATGGGACTATGTGAGC	TTTGCCGTGTGGAAAAACCG
	F5 Gusb Hamp Heph Hfe Hjv Hprt1 Slc11a2 Slc31a1 Slc39a14 Slc40a1	ATCTATGGGAGGAGGCACGA TCGAACAATCGGTTGCAGG CTGCAGCCTTGGCATGG ATGCACTGCCACGTTACTGA GTGGGATCACCATTTGTGCCAT TCCTCTTTGTCCAAGCCACC CTGATTATGGACAGGACTGAAAGAC CTTTGTCGTCTCCGTCTTTGC ACGAGATGATGATGCCTATGACC GGCTGGAGGATTTCAGTGTGT AGGGACTGGATTGTTGTCGT M a n u s c r i p t TGCGTCTTGGATTGGAACTCA AGCCAATGAAGTTCCGAAGC CAGCAGCGCACTGTCATCA CTGCAGAATCACAGCTTTTCCA ATGCTGCAGGTCACTCACACT TCGCCCCCATTGACAGAAC CCAGCAGGTCAGCAAAGAACT ATGGGGGCTGCTGCTATTTC AACACTGCCACAAAAGCTCC ACCAAACAGCACCAACAGGA GACTGGGGAACCGAATGTCA
	Tbp	GGGATTGTACCACAGCTCCA	CAGCAAACCGCTTGGGATTA
	Tfrc Tfr2 Supplemental data A c c e p t e d AGCTGGACTGCAGGAGACTA CATGTACAACGTGCGCATCA	GGGCTGGCAGAAACCTTGAA GGAAAATGTGGCGGAATGGG

Table 2 :

 2 Metal concentrations in liver, spleen, plasma, and erythrocytes. ns : no statistically significant difference.

Acknowledgements

This work was supported by the "Fondation maladies rares", the "Région Bretagne" (MK), INSERM and AFeMER. This work was partially supported by the "TEFOR" project funded by the « Investissements d'Avenir » French Government program (ANRII-INSB-0014), and also member of "CELPHEDIA" infrastructure. This work was realized in the context of the Labex IGO project (ANR-11-LABX-0016-01) and the IHU-Cesti project (ANR-10-IBHU-005) which both are part of the « Investissements d'Avenir » French Government program. The IHU-Cesti project is also supported by Nantes Métropole and Région Pays de la Loire. The author's thanks the U1154-UMR7196 TACGENE for design and produce the sgRNA.

Abbreviations

Cp, ceruloplasmin ; HA, hereditary aceruloplasminemia ; Hamp, hepcidin antimicrobial peptide ; Heph, hephaestin ; Hjv, hemojuvelin BMP co-receptor ; LIC, liver iron concentration ; NTBI, non-transferrin bound iron ; SIC, spleen iron concentration ; Slc11a2, solute carrier family 11 member 2 ; Slc39a14, solute carrier family 39 member 14 ; Slc40a1, solute carrier family 40 member 1 ; Tfrc, transferrin receptor ; Tfr2, transferrin receptor 2 ; UIBC, unsaturated iron bound capacity.


Vincent Placet
email: vincent.placet@univ-fcomte.fr

Mélissa Blot

Tine Weemaes

Hugo Bernollin

Guillaume Laurent

Fabien Amiot

Cédric Clévy

Johnny Beaugrand

TRANSNVERSE COMPRESSIVE PROPERTIES OF NATURAL FIBRES DETERMINED USING MICRO MECHATRONICS SYSTEMS AND 2D FULL-FIELD MEASUREMENTS

published or not. The documents may come

Transverse compressive properties of natural fibres determined using micro mechatronics systems and 2D full -field measurements

Vincent Placet, Mélissa Blot, Tine Weemaes, Hugo Bernollin, Guillaume Laurent, Fabien Amiot, Cédric Clevy, Johnny Beaugrand

To cite this version:

INTRODUCTION

One of the main shortcomings of current knowledge on plant fibres is related to their transverse mechanical properties. To the best of the author's knowledge, no data determined by direct methods can be found in the open literature. Only values determined by backcalculation from tensile tests realised in the transverse direction of unidirectional composite materials [START_REF] Baley | Transverse tensile behaviour of unidirectional plies reinforced with flax fibres[END_REF] are available. More generally, literature on the transverse mechanical properties of synthetic monofilaments is sparse compared with the voluminous data on their longitudinal properties. In this study, we propose to develop an experimental device and methodology to accurately determine the transverse compressive properties of polymeric monofilaments and plant fibres. This compression experimental test is commonly called as « Brazilian » test or as Fibre Transverse Compression Test (FTCT) and has been proposed and developed since the 60's [START_REF] Hadley | The transverse compression of anisotropic fibre monofilaments[END_REF]. It has been mainly applied on single synthetic fibres and textile fibres assemblies, so far. In most of the works, the fibres and monofilaments are assumed as perfectly cylindrical and made of elastic isotropic of transversely isotropic material. The problem is treated as for two semi-infinite solids in contact (Hertz) under conditions of plane strain. Literature points out that conducting such experiments on small diameter polymeric fibres is really challenging. Indeed, their small diameter and low modulus make the test specimen very compliant per length unit. The tests require also an accurate alignment and measurement of platen displacement. In addition, this experiment is very sensitive to the geometry of the contact between fibre and platen and consequently on variations in the fibre diameter. The transverse compressive modulus is generally identified by inverse method using analytical models [START_REF] Jawad | The transverse compression of oriented nylon and polyethylene extrudates[END_REF]. For anisotropic materials, the model is sensitive to the other elastic properties (longitudinal elastic modulus and major Poisson's ratio). To cope with these difficulties, a specific methodology and the associated device were developed. It comprises a set-up based on micro mechatronics system and 2D full-field strain measurements at the microscale. The longitudinal elastic modulus and the major Poisson's ratio are first determined using single fibre tensile tests. The transverse elastic modulus is then determined from the FTCT and more exactly from the measured compressive load, the 2D full field displacement and the Kolosov-Muskhelishvili formulation.

RESULTS AND CONCLUSIONS

Fig. 1 shows some pictures of the developed set-up, the transverse cross-section of a PA11 monofilament tested, and the measured load-displacement curve.

Fig. 1

 1 Fig.1 Transverse compressive set-up and results obtained on Pa11 monofilament The first tests were performed on Polyamide 11 (PA11) monofilaments, with an average diameter of approximately 40 m. The elastic longitudinal modulus, determined using single fibre tensile tests, is equal to approximately 2100 MPa. The transverse compressive elastic modulus identified from the analysis of the 2D full field displacement field recorded during compression tests is approximately 1450 MPa. The presentation will also include preliminary results obtained on plant and animal fibers.

th ICNF -Smart Sustainable Solutions 1

ACKNOWLEDGMENTS

The authors gratefully acknowledge the financial support provided by the 'ANS μReshape' received from the CEPIA Science for Food and Bioproduct engineering department of INRA.