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Introduction

Mastitis and metritis are considered as two major concerns in dairy production. Indeed, frequency, high economic losses [START_REF] Sheldon | Defining Postpartum Uterine Disease and the Mechanisms of Infection and Immunity in the Female Reproductive Tract in Cattle1[END_REF][START_REF] Steeneveld | Cow-specific treatment of clinical mastitis : An economic approach[END_REF], impact on animal welfare (Fthenakis and Jones, 1990a ;[START_REF] Gougoulis | Subclinical mastitis changes the patterns of maternal-offspring behaviour in dairy sheep[END_REF] , quality and safety of animal products (Fthenakis and Jones, 1990b) and overconsumption of antibiotics make these two diseases the most damaging infectious diseases in dairy farming. Furthermore, mastitis and metritis are both due to bacterial infections and appear more often during the first 3 weeks postpartum in dairy cattle and sheep [START_REF] Mallard | Alteration in Immune Responsiveness During the Peripartum Period and Its Ramification on Dairy Cow and Calf Health[END_REF][START_REF] Pomeroy | Counts of bovine monocyte subsets prior to calving are predictive for postpartum occurrence of mastitis and metritis[END_REF]. These infections are thought to be due to a sub-optimal immune response during the periparturient period which predisposes ruminants to several metabolic and productionrelated health problems [START_REF] Goff | Physiological Changes at Parturition and Their Relationship to Metabolic Disorders[END_REF]. However, for decades, dairy cattle selection objectives have centred on milk production, with limited attention being given to traits such as health and fertility. Geneticists have observed negative genetic correlations between milk production traits, as well as reproduction and health traits [START_REF] Pryce | Fertility in the high-producing dairy cow[END_REF] including resistance to mastitis and metritis [START_REF] Heringstad | Selection Responses for Clinical Mastitis and Protein Yield in Two Norwegian Dairy Cattle Selection Experiments[END_REF][START_REF] Oltenacu | Selection for Increased Production and the Welfare of Dairy Cows : Are New Breeding Goals Needed ?[END_REF].

Therefore genetic selection for milk production in dairy ruminants is moving forward to take into consideration both production efficiency and resistance to uterine and mammary infections.

Metritis is a uterine microbial disease which affects 40% of post-partum cows, causing infertility by disrupting uterine and ovarian function [START_REF] Borsberry | Periparturient diseases and their effect on reproductive performance in five dairy herds[END_REF][START_REF] Opsomer | Risk factors for post partum ovarian dysfunction in high producing dairy cows in Belgium : A field study[END_REF]. It occurs within 21 days after parturition and is characterized by an enlarged uterus and a watery red-brown fluid to viscous off-white purulent uterine discharge [START_REF] Sheldon | Defining postpartum uterine disease in cattle[END_REF]. For ruminants whith persistent infection, development of clinical endometritis is defined as the presence of a purulent uterine discharge detectable in the vagina 21 days or more post-partum. Subclinical endometritis is characterised by inflammation of the endometrium in the absence of signs of clinical endometritis [START_REF] Sheldon | Defining Postpartum Uterine Disease and the Mechanisms of Infection and Immunity in the Female Reproductive Tract in Cattle1[END_REF]. In bovine endometrial epithelial cells, a 24h challenge with Escherichia coli is associated with an increased expression of innate immunity related genes including Toll-like receptor 4 (TLR4), the transcription factor NFkB1, the chemokine interleukin 8 (IL-8), inflammatory cytokines (interleukins IL-1, IL-6, tumour necrosis factor TNF), defensins (lingual antimicrobial peptides LAP, tracheal antimicrobial peptide TAP) and acute phase proteins (haptoglobin HP, serum amyloid A SAA3) [START_REF] Chapwanya | Endometrial epithelial cells are potent producers of tracheal antimicrobial peptide and serum amyloid A3 gene expression in response to E. coli stimulation[END_REF]. Furthermore, in vivo study with Analysing bovine endometrial biopsies by Next-Generation Sequencing, at 21 days post-partum, has shown 1167 significantly differentially genes between healthy cows and cows diagnosed with clinical endometritis including cell surface receptors (the TNFreceptor superfamily member CD27, the calcium signalling receptor CD38, B-cell receptors CD79A and CD79B), chemokine ligands and receptors (CCL20, CCL22, CCL24 and members of the CXC family as well as CXCR2), cytokines and receptors (IL1R2, IL6, IL10 and IL11), antimicrobial peptides (DEFB5, DEFB7, LAP and multiple members of the S100A gene family) all being are up-regulated in clinical endometritis samples. Therefore, all these immune genes are worth to be examined as response patterns to uterine infections in dairy ruminants.

Mastitis is an infectious disease characterised by an inflammation of the mammary gland, which in its severe form causes abnormal milk and changes in the udder with swelling, heat, pain and redness. Inflammation of the mammary gland and chronic mastitis are detected thanks to milk Somatic Cell Count (SCC) which reflects the number of neutrophils that migrate from blood to the mammary gland in response to an intra-mammary infection. To achieve data normality, SCC are commonly log-transformed to Somatic Cell Score (SCS) [START_REF] Ali | An Optimum Transformation for Somatic Cell Concentration in Milk[END_REF] and could be use in selection with a heritability of 0.15 in dairy cattle (Rupp and Boichard, 2003). Research teams of Gilles Foucras (IHAP, INRA-ENVT, Toulouse, France) and Rachel Rupp (GenPhySE, INRA, Toulouse, France) have amplified two ovine lines : Low SCS (LSCS) with (wild type SOCS2 ) genotype and High SCS (HSCS) with (mutant SOCS2 ) genotype in order to study the long-term effects and efficacy of sole-SCS selection and its consequences on the resistance of the host to intra-mammary infections. A Single Nucleotide Polymorphism (SNP) mapping to the coding sequence of the suppressor of cytokine signaling 2 (SOCS2) gene was described in these ovine lines with a higher frequency in HSCS ewes [START_REF] Rupp | A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model[END_REF]. To control this variability, a selection by SOCS2 genotype was performed. Furthermore, the ovine species has been chosen as a model for studying ruminant mastitis because of the better feasibility of breeding a significant number of animals, the availability of SCS recording and genetic evaluation on a large sheep population, and strong similarities in the physiopathology of mastitis with dairy cattle. Results from experiments carried out on mastitis resistance in dairy sheep can be readily transposed to dairy cattle [START_REF] Rupp | Response to somatic cell count-based selection for mastitis resistance in a divergent selection experiment in sheep[END_REF]. Studies on these ovine lines demonstrated that bacterial clearance is more efficient in the LSCS ewes than in the HSCS ewes after experimental intramammary infection [START_REF] Bonnefont | Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus[END_REF]. Consequently, the LSCS line is regarded as resistant and the HSCS line as susceptible to mastitis.

In this context, we have hypothesized that divergent susceptibility to mastitis could be associated with a divergent ability to cope with uterine infection. HSCS and LSCS lines were selected as models for this study for breeding reasons and because uterine post-partum infections and acute endometritis in ewes is much less common than in cattle [START_REF] Palmieri | Congenital and acquired pathology of ovary and tubular genital organs in ewes : A review[END_REF] that allow experiments with a healthy uterine environment. To generate a comprehensive and integrative view of the endometrial transcriptome response to an experimental infection, we analyzed caruncular (CAR) and intercaruncular (ICAR) separately. Indeed, the endometrium of ruminants have a bicornuate uterus with a small common corpus. The endometrium of ruminants, which is the inner epithelial layer of the uterus, has a large number of aglandular caruncular areas (CAR), which are dense stromal areas covered by a simple luminal epithelium, and intercaruncular areas (ICAR) that contain many hundreds of glands per cross-section of the uterine wall (Spencer, 2014). This work reports the transcriptomic analyses of this two CAR and ICAR endometrial areas collected from mastitis resistant and mastitis susceptible ewes that were experimentally infected by intra-uterine infusion of E. coli.

Materials and Methods

Animals, experimental challenges and sample collection

Twenty-four multiparous and lactating Lacaune ewes (INRA experimental facility of La Fage, UE321, Roquefort, France) were used for this study. They belonged to two genetic lines divergently selected since 2003 for extreme milk SCS : low and high somatic cell score lines, respectively LSCS and HSCS [START_REF] Rupp | Response to somatic cell count-based selection for mastitis resistance in a divergent selection experiment in sheep[END_REF]. Previous studies had shown that LSCS ewes are characterized by higher resistance to mastitis than HSCS ewes [START_REF] Rupp | Response to somatic cell count-based selection for mastitis resistance in a divergent selection experiment in sheep[END_REF].

Ewes from each genetic background were housed together in the same facility and managed identically. In order to avoid bias, animals were estrus synchronized for 9 days using a CRESTAR progestagen implant (Intervet) associated with injection of 2mL of cloprostenol, a prostaglandin analogue, 2 days before bacterial infusion. Experimental endometritis was induced by the infusion of E. coli MS499 previously isolated from Holstein cattle with the postpartum disease metritis [START_REF] Goldstone | Draft Genome Sequence of Escherichia coli MS499, Isolated from the Infected Uterus of a Postpartum Cow with Metritis[END_REF][START_REF] Sheldon | Specific Strains of Escherichia coli Are Pathogenic for the Endometrium of Cattle and Cause Pelvic Inflammatory Disease in Cattle and Mice[END_REF]. The inoculum was prepared so that the number of bacteria in the inocula was 7-8x10 7 colony-forming units/mL. Twelve hours after progestogen implant removal, 5mL of bacterial suspension were injected into each uterine horn using the miniaturized embryo transfer syringe and sheath (IMV) under general anesthesia [START_REF] Constant | Large Offspring or Large Placenta Syndrome ? Morphometric Analysis of Late Gestation Bovine Placentomes from Somatic Nuclear Transfer Pregnancies Complicated by Hydrallantois[END_REF]. Twenty four hours after challenge, ewes were slaughtered then the uteri were collected and flushed with PBS (10mL/horn). The uterine horns were then dissected in order to remove caruncular (CAR) and intercaroncular (ICAR) areas separately by the same experimentator. All samples were immediately placed into RNAlater in order to be transported to the host laboratory. Forty eight hours after sampling, RNAlater was removed then endometrial samples were stored at -80 • until further processed.

Experimental design

A first subgroup of twelve ewes was slaughtered in April 2016 and used to perform a Next-Generation Sequencing (NGS) of mRNA from endometrial tissues. Four ewes from the LSCS line with the wild type SOCS2 genotype constitute a control group which was inoculated with 5mL PBS only (Control LSCS). An experimental group of five LSCS (wild type SOCS2) animals were inoculated with 5mL of E. coli infusion per horn (Inoculated LSCS) and five ewes from the HSCS line (mutant SOCS2) genotype were also inoculated with 5mL of E. coli infusion per horn (Inoculated HSCS). One [Inoculated LSCS] ewe was removed from the experiment because the bacterial concentration of its uterine flush was more similar to those from [Inoculated HSCE] ewes. One [Inoculated HSCE] ewe was also removed from the experiment for identical reasons. A second subgroup of ten ewes was slaughtered in September 2016 in order to increase the number of samples for validation of NGS analyses. Five ewes from the LSCS line (wild type SOCS2) and five ewes from the HSCS line and (mutant SOCS2) were inoculated with 5mL of E. coli infusion per horn.

RNA extraction, library preparation and NGS sequencing

Samples were homogenized in TRIzol (Invitrogen) using an Ultra-Turrax R disperser (Sigma-Aldrich) and total RNA was isolated using the RNeasy Min Kit (Qiagen) following Chomczynski and Sacchi protocol [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF]. Quality and integrity of purified total RNA were determined by Agilent 2100 BioAnalyzer. The RIN index was ranged from 5.1 to 9.2 and samples with a RIN above 7 were selected for sequencing, namely Next Generation Sequencing data repository [START_REF] Mariette | NG6 : Integrated next generation sequencing storage and processing environment[END_REF] hosted by GenoToul (http ://ng6.toulouse.inra.fr/).

Quality control and alignment of reads

The quality control check of the raw reads was performed with the FastQC software (version 0.11.2) (http ://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The used assessments included per base sequence quality, per sequence quality, GC content, Kmer content, sequence length distribution, per base N count and overrepresented sequences.

Adapter sequences were removed from sequencing reads with Cutadapt command-line program (version 1.8.3) [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF] and the quality of reads was checked a second time with FastQC software. Reads were then mapped to the ovine genome (Ovis aries version 3.1.87) using the splice junction mapper TopHat2 (version 2.0.14) [START_REF] Kim | TopHat2 : accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions[END_REF] associated with short read aligner Bowtie2 (version 2.1.0) [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]. Finally, featureCounts (version 1.4.5) [START_REF] Liao | featureCounts : an efficient general purpose program for assigning sequence reads to genomic features[END_REF] was used to summarize at the gene level the number of aligned reads per exon using the Ensembl annotation of the ovine genome. The featureCounts multi-mapping (-m) option was applied to count every alignment reported for a multi-mapping read i.e. reads mapping to more than one genomic location with the same similarity score. Ensembl gene IDs were used in the output file to identify the number of read counts per gene.

Gene expression data analysis and statistics

Differentially expressed genes (DEGs) were identified using the Bioconductor DESeq2 package version 1.14.1 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. Prior to statistical analyses, read count data were filtered and transcripts with less than 10 reads for all samples were removed (very low expression). Gene counts were normalized using the regularized log transformation procedure provided by DESeq2. Principal Component Analysis (PCA) and Hierarchical Clustering (HC) were performed using the FactoMineR package [START_REF] Lê | FactoMineR : An R Package for Multivariate Analysis[END_REF] to check the dispersion estimates of the individual samples using the normalized counts of all genes.

Analyses among to identify DEGs were performed separately for CAR and ICAR samples and the Benjamini-Hochberg false discovery rate (FDR) was used in the determination of DE expression with an adjusted p-value < 0.05.

Enrichment of genes within GO terms and associated biological pathways

Ovine gene IDs retained were converted to human orthologs using the BioMart application available on the Ensembl website (http ://www.ensembl.org/biomart/martview/).

In order to form series of clusters with genes grouped according to their biological function, significant genes from the DE analysis were submitted to the Database for Annotation, Visualization and Integrated Discovery (DAVID version 6.8) with human ortholog gene names (HUGO nomenclature) to perform a functional annotation clustering [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF] with Gene Ontology : Biological Process terms and KEGG canonical pathways dataset.

Clusters were considered significant if Enrichment Score (ES) > 1.3, which is equivalent to non-log scale 0.05. Iside each cluster, GO terms were retained if FDR<0.05. Thereafter, annotations terms for each cluster were summarize into one GO term thanks to ReViGO, a web server that summarizes lists of GO terms by finding a representative subset of the terms thanks to their p-values in this case [START_REF] Supek | REVIGO Summarizes and Visualizes Long Lists of Gene Ontology Terms[END_REF].

A gene set enrichment analysis (GSEA) was performed using the software available (version 2.0) at the Broad Institute Gene Set Enrichment Analysis website. GSEA is a computational method that determines whether a defined set of genes shows statistically significant, concordant differences between two states or groups of individuals thanks to their genomewide expression profiles [START_REF] Subramanian | Gene set enrichment analysis : A knowledge-based approach for interpreting genome-wide expression profiles[END_REF]. We estimated the gene's differential expression with respect to the two phenotypes thanks to the signal-to-noise ratio (difference of means scaled by the standard deviation) and statistical significance of the enrichments score was assessed by performing 1000 gene-set permutations. Pathways gene sets tested were Gene Ontology Biological Process terms, KEGG pathways gene set and Hallmark gene set. The latter was generated by a computational methodology based on identifying overlaps between gene sets in other gene collections and retaining genes that display coordinate expression and summarizes specific well-defined biological states or processes and display coherent expression. Since gene permutations parameter was used, gene sets were considered as differentially expressed if FDR<0.05. All these procedures were applied separately for each comparison.

Candidate genes selection and validation of gene expression by qPCR

Validation of DEGs identified by RNA-seq are currently under investigation with a selection of forty two candidate genes. These genes were selected from RNA-seq DEGs list based on a combination of criteria including : FDR<0.05, |log2FC|>1, read counts, pathway analysis and relevance in accordance with the study. Primers for qPCR were designed using PRIMER-Blast website (https ://www.ncbi.nlm.nih.gov/ tools/primer-blast/) and synthesised commercially by Eurofins Genomics (Ebersberg, Germany). The specificity of each designed primers was checked with BLAST (http ://blast.ncbi. nlm.nih.gov/). The high-throughput microfluidic BioMark qPCR platform (Fluidigm) will be used for qPCR analyses and will be performed by GeT-PlaGe (GenoToul, Toulouse, France).

Results

Statistical analyses of expression profiles

Principal Component Analysis (PCA) of all samples reveals that the first principal component, which explains 23.66% of the total variation, discriminates CAR samples from ICAR samples (Figure 1-A). PCA for CAR samples shows, with the two first principal components (46.92%), a separation by genotype. Nevertheless, this observation is limited by the few

FIGURE 3 -GO biological process categories in DEGs for [Inoculated LSCS] vs [Control LSCS]

PIE CHART : Gene Ontology (GO) clusters from DAVID Functionnal Annotation Clustering were grouped according to their associated biological processes.

GSEA analysis of [Inoculated LSCS] vs [Control LSCS]

GSEA analysis was run to identify pathways with numerous differentially expressed genes using genomewide expression profiles and considering the contribution of genes with a small degree of expression difference in a defined gene set. GSEA results show that up-regulated gene sets in [Control LSCS] ewes include protein and amino acid metabolism processes (Ribosome, Tetrapyrrole metabolic process) whereas significative over-expressed pathways in [Inoculated LSCS] ewes are involved in recognition of pathogens (NOD-like, RIG-I-like and Toll-like receptors signaling), acute phase response (IL6-JAK-STAT3 signaling), cytokine production, sex-hormones response (Androgen response and Estrogen response early), innate immunity (Complement) and generation of cell-mediated immunity (Interferon gamma and alpha response) but not humoral immunity (Tables 12).

GSEA analysis of [Inoculated LSCS] vs [Inoculated HSCS]

GSEA analysis was performed for CAR and ICAR samples distinctly. Results show similar differentially expressed pathways in CAR and ICAR areas, therefore interpretations of GSEA results from both tissues have been merge.

Between the two lines of ewes, a significant difference in the number of pathways differentially expressed is observed with 10 times more up-regulated pathways for inoculated ewes and 20 to 50 times more enriched GO biological process terms (Table 3). (Antigen processing and presentation, Leukocyte mediated immunity) and in disorders of the immune system causing increased susceptibility to infection (Primary immunodeficiency).

[Inoculated LSCS] ewes do not show many over-expressed pathways. None of them is linked to immunity but relates to cellular morphogenesis processes (Cilium organization, Aminoglycan biosynthetic process) (Table 4). 5).

Discussion

Bacterial uterine infections are a major problem in dairy production because they compromise animal welfare, increase costs of therapeutic treatment, reduce milk production and lead to the replacement of infertile animals. The economic cost of a single case of metritis has been estimated to be about e292 per dairy cow in EU [START_REF] Drillich | Evaluation of a Systemic Antibiotic Treatment of Toxic Puerperal Metritis in Dairy Cows[END_REF]. Consequently, genetic selection of dairy ruminants has to consider resistance to uterine disease. In the present study, the biological endometrial response to an acute experimental uterine infection was assessed, using two lines of Lacaune dairy sheep selected for their divergent ability to cope with mastitis.

The effects of an experimental uterine infection on endometrial reaction were investigated using endometrial tissues collected from [Control LSCS] and [Inoculated LSCS] ewes. In ICAR areas, transcriptional expression levels of 99 genes were found significantly altered in the endometrium of ewes infected compared to the control group. These DEGs are mainly involved in inflammation response, in innate immunity and in cell-mediated immunity but not humoral immunity, including genes encoding chemokine ligands and receptors (CCL2, CCL8, CCL20, CXCL2, CXCL3, CXCL10, CXCR2), cytokines (IL-1α, IL-1β, IL-8) and antimicrobial peptides (S100A9, LYZ). IL-1 and IL-8 are pro-inflammatory cytokines which are secreted by bovine endometrial stromal and epithelial cells in response to LPS [START_REF] Herath | Expression and Function of Toll-Like Receptor 4 in the Endometrial Cells of the Uterus[END_REF], and by endometrial explants in response to Gram-negative or Gram-positive bacteria [START_REF] Borges | Explants of Intact Endometrium to Model Bovine Innate Immunity and Inflammation Ex Vivo[END_REF]. These two cytokines are detected in endometrial biopsies from lactating dairy cows diagnosed with metritis [START_REF] Kasimanickam | Mucin 1 and cytokines mRNA in endometrium of dairy cows with postpartum uterine disease or repeat breeding[END_REF]. IL-1 is a mediator of the host's inflammatory response to infections and a high concentration of IL-1 is thought to influence the systemic inflammatory response to induce APP synthesis in the liver, and to stimulate IL-6 secretion and the production of neutrophils in the bone marrow [START_REF] Burger | Cytokines, acute-phase proteins, and hormones : IL-1 and TNF-alpha production in contactmediated activation of monocytes by T lymphocytes[END_REF]. IL-8 is a chemokine that attracts immune cells, particularly neutrophils and a high expression of IL-8 with accumulation of neutrophils can lead to the formation of pus [START_REF] Carneiro | Mechanisms linking bacterial infections of the bovine endometrium to disease and infertility[END_REF]. Neutrophils are a rich source of natural anti-microbial molecules including defensins, lactoferrin and lysozyme [START_REF] Ganz | Defensins. Natural peptide antibiotics of human neutrophils[END_REF]. The coding gene for lysozyme, LYZ, was also found to be increased in the endometrium of cows with uterine inflammation [START_REF] Hoelker | Transcriptional response of the bovine endometrium and embryo to endometrial polymorphonuclear neutrophil infiltration as an indicator of subclinical inflammation of the uterine environment[END_REF]. The antimicrobial role of this peptide is to digest the peptidoglycans found in the cell walls of bacteria. Neutrophils also contribute to bacterial clearance by producing reactive oxygen species (ROS). ROS production is critical for the degradation of internalized bacteria but it can also contribute to inflammatory damage of host tissue if unbalanced [START_REF] Chen | Measurement of Oxidative Burst in Neutrophils[END_REF]. Interestingly, SOD1 and SOD2 transcripts were found to be up-regulated in [Inoculated LSCS] ewes. SOD proteins are superoxide dismutase isozymes responsible for destroying ROS and balancing ROS production. Altogether, in reply to the experimental infection in LSCS ovine line, our data confirm an endometrial acute phase response that is consistent with published data related to uterine infection in cattle.

Analysing CAR and ICAR endometrial areas independently appears relevant to generate a comprehensive and integrative view of the endometrial transcriptome response to an experimental infection and to the best of our knowledge, such an analysis has never been reported. Our data underline that the impact of the 24h experimental infection between LSCS and HSCS lines was 3 times more prominent in the ICAR areas, considering the higher number of DEGs. CAR areas are dense stroma protuberances covered by luminal epithelium and match the sites of implantation and placentation, whereas ICAR areas contain large numbers of endometrial glands which produce a variety of molecules, including enzymes, cytokines, growth factors, ions, hormones, glucose, transport proteins, and adhesion molecules, collectively referred to as histotroph, that are required for conceptus survival and development [START_REF] Gray | Endometrial Glands Are Required for Preimplantation Conceptus Elongation and Survival1[END_REF]. Common DEGs between CAR and ICAR areas were found and are mainly involved in innate and cell-mediated adaptive immune responses.

We observed that altered specific biological functions in CAR areas appeared to be mainly related to intracellular signal transduction unlike in ICAR areas for which modified biological functions mainly relate to to metabolism processes and biogenesis. Enrichment in biogenesis processes in ICAR areas is likely related to the secretory activity of endometrial glands, specifically found in this endometrial zone. Furthermore, vascularisation of the endometrium is mainly found close to endometrial glands. Thus it is likely that immune cell recruitment in response to the infection takes place preferably in ICAR areas. Therefore, to better understand the differential response between CAR and ICAR areas, evaluating endometrial gland density and identifying immune cell types in each endometrial area is necessary.

In addition, a CAR vs ICAR comparison of gene expression profiles should be carried out using a linear mixed model : "line + line/ewe + tissue". This model takes into consideration (i) the affiliation between CAR and ICAR areas samples from the same ewe (ewe effect), (ii) the fact that ewes belong to different lines (line effect) thanks to the "line/ewe" term (ewe and line nested effects), and (iii) the random effect due to a randomly sampling of ewes from a population. Unfortunately, in the present study the low numbers of paired CAR and ICAR samples (3 LSCS CAR-CAR pairs and 4 HSCS CAR-ICAR pairs) that were sequenced has prevented us from running a relevant analysis. Additional samples will be sequenced for making such an analysis possible.

The major objective of this study was to assess the biological endometrial response to an uterine infection in two lines of dairy sheep : LSCS ewes, which are resistant to mastitis, and HSCS ewes, which are susceptible to mastitis. Thanks to our exploratory data, we have confirmed that expression profiles of all samples cluster according to the line and the treatment, even though individuals were more segregate by line than by treatment. In HSCS and LSCS lines upon intra-mammary Staphylococcus epidermidis and S. aureus infection, a transcriptomic analysis of milk somatic cells has been performed and shows some similar results with our study [START_REF] Bonnefont | Transcriptomic analysis of milk somatic cells in mastitis resistant and susceptible sheep upon challenge with Staphylococcus epidermidis and Staphylococcus aureus[END_REF]. No expression differences wes detected for major chemokines such as IL-1β, TNFα, IL-6 and IL-8 , that up-regulated genes in HSCS line were involved in the stimulation of inflammatory response and that genes coding for acute phase proteins (SAA2), antibacterial proteins (S100A2) and chemokine C-C motif ligands (CCL5) were significantly more expressed in the HSCS line. However, bacteria titres were higher in the LSCS line than in the HSCS line 12h after the intra-mammary infection but more neutrophils were quantified in mammary glands of HSCS line. Bonnefont et coll. (2011) conclude that HSCS neutrophils were less efficient in killing Staphylococcus. Resistance of LSCS line to mastitis should be explained by a more efficient pathogen recognition through the TLR2 signaling pathway and by an enhanced cell turn-over. In our study, with the same ovine lines, more neutrophils were quantified in the uterine lumen of HSCS line but bacteria titres were higher in the LSCS line than in the HSCS line 24h after endometrial infection. We can explain this paradox by the use of different pathogens : Staphylococcus epidermidis and aureus (Gram-positive bacteria) for the study of resistance to mastitis and Escherichia coli (Gram-negative bacteria) for the study of resistance to metritis. Indeed, neutrophils do not have the same efficiency according to the pathogen and some bacteria, such as Staphylococcus aureas, have the ability to overcome neutrophil-mediated host defense by thwarting neutrophil chemotaxis and phagocytosis [START_REF] Teng | Neutrophils and Immunity : From Bactericidal Action to Being Conquered[END_REF]. By comparing our data with Bonnefont et coll. ( 2011), we could suggest that distinct immune mechanisms are involved in resistance to mastitis and resistance to metritis.

In dairy cows during the postpartum period, two patterns of immune and inflammatory response have been reported : "Excessive" inflammation of the endometrium which refers to inflammation that is severe or prolonged enough to impair rather than aid reproductive performance and "Inadequate response" of the endometrium which refers to innate immune function that fails to clear uterine pathogens within 3 weeks after calving [START_REF] Leblanc | Reproductive tract inflammatory disease in postpartum dairy cows[END_REF].

In this study, 24h after the bacterial challenge, we observe a better clearance of the uterine lumen, a higher number of neutrophils and a transcriptomic profil enriched in DEGs involved in immune response processes for mastitis susceptible ewes, HSCS line. Therefore, two main hypothesis can be suggested : (i) Selection of mastitis resistance in LSCS line leads to counterselection of metritis susceptibility or (ii) HSCS line shows an intense inflammatory response due to an excessive recruitment of neutrophils leading to tissue wounding and susceptibility to chronic metritis.

Several of our results are in favor of the second hypothesis. First, GSEA analyses showed that the Primary Immunodeficiency (PIDD) KEGG pathway was enriched in [Inoculated HSCS] ewes. PIDDs are disorders divided into nine groups whose autoinflammatory disorders [START_REF] Ochs | Primary immunodeficiency disorders : general classification, new molecular insights, and practical approach to diagnosis and treatment[END_REF]. Secondly, a Quantitative Trait Loci (QTL) associated with SCS index was recently identified and fine mapping of the region provided a strong candidate : a Single Nucleotide Polymorphism (SNP) mapping to the coding sequence of the suppressor of cytokine signaling 2 (SOCS2) gene [START_REF] Rupp | A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model[END_REF]. SOCS proteins act as negative feedback factors of cytokine, growth factors and hormones action by inhibiting intracellular signaling pathways, including the JAK/ STAT pathway. Interestingly, SOCS2 deficient mice infected with Toxoplasma gondii have been shown to have uncontrolled production of proinflammatory cytokines, decreased microbial proliferation, aberrant leukocyte infiltration and elevated mortality [START_REF] Machado | Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent[END_REF]. In our study, HSCS ewes were selected with a mutant SOCS2 genotype, i.e. these animals were heterozygous carriers of the identified SNP on SOCS2 gene. This mutation causes an amino-acid change of SOCS2 protein comparable to a functional knock-out of SOCS2. This may explain that HSCS ewes selected for mutant A positive or negative association between mastitis resistance and metritis resistance will require additional analyses.

Résumé

Les mammites et métrites sont des maladies inflammatoires fréquentes en élevage laitier, responsables de pertes économiques élevées, d'une altération de la santé et du bien-être des animaux.

Une meilleure maîtrise de ces infections peut être envisagée par, notamment, la prise en compte de l'impact du fond génétique des animaux sur la réponse aux maladies inflammatoires. Ce projet a pour objectif d'évaluer si la sélection génétique pour une meilleure résistance aux mammites modifie la sensibilité aux infections utérines.

Cette étude repose sur l'utilisation de deux lignées de brebis sélectionnées pour des index extrêmes de [HSCS] ont été sélectionnées pour un génotype SOCS2 mutant qui équivaut à une perte de la fonction biologique de la protéine [START_REF] Rupp | A Point Mutation in Suppressor of Cytokine Signalling 2 (Socs2) Increases the Susceptibility to Inflammation of the Mammary Gland while Associated with Higher Body Weight and Size and Higher Milk Production in a Sheep Model[END_REF]. Or, il a été observé que des souris déficientes pour SOCS2 présentaient, en réponse à une infection, un recrutement excessifs des cellules immunitaires et une sur-inflammation des tissus [START_REF] Machado | Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent[END_REF]. Enfin, l'analyse histologique des endomètres a mis en évidence un recrutement des neutrophiles plus important pour la lignée [HSCS] associée à la formation d'oedèmes. Des analyses complémentaires sur les effets à plus long terme de l'infection sont nécessaire pour conclure sur la récupération fonctionnelle du tissu endométrial de la lignée

[HSCS] et sur l'efficacité de la réponse immunitaire de la lignée [LSCS] afin de déterminer si cette réaction différentielle aux infections utérines est, ou non, liée à une préservation de la fonction endométriale nécessaire pour le bon déroulement de la gestation.

  Abbreviations

  10 CAR samples (2 [Control LSCS] + 4 [Inoculated LSCS] + 4 [Inoculated HSCS]) and 12 ICAR samples (4 [Control LSCS] + 4 [Inoculated LSCS] + 4 [Inoculated HSCS]). Sequencing of 150 bp reads paired-end cDNA libraries, obtained after fragmentation, retro-transcription and amplification of total RNA, was performed with an Illumina R HiSeq TM 3000 and a depth of 25X (GeT-PlaGe, GenoToul, Toulouse, France). Raw data were stored in nG6, a

[

  Control LSCS] individuals (Figure 1-B). Similarly, PCA of ICAR samples shows, with the first principal component (29.95%), a clear separation by genotype and, with the second (21.17%), a discrimination between inoculated and non-inoculated ewes (Figure 1-C). Hierachical clusterings (HC) reinforce these results and confirm that resistant and susceptible lines are separated, regardless of the challenge.

FIGURE 1 -

 1 FIGURE 1 -Principal Component Analysis and Hierarchical Clustering of all samples A : PCA and HC of all samples -B : PCA and HC of CAR samples -C : PCA and HC of ICAR samples -Samples were labelled RU# which is an experimental identification. HC were calculated with Ward's method and Euclidean distance.

FIGURE 2 -

 2 FIGURE 2 -Volcano-plot and top 10 DEGs between [Inoculated LSCS] and [Control LSCS] VOLCANO-PLOT : Orange dots are up-regulated DEGs in [Inoculated LSCS] ewes with absolute log2(FC)>2 and FDR<0.05. Green dots are up-regulated DEGs in [Control LSCS] ewes with absolute log2(FC)>2 and FDR<0.05. The 10 shown DEGs are the 5 DEGs with the highest FC and the 5 DEGs with the lowest FC. TABLE : The 10 most significant DEGs between [Inoculated LSCS] and [Control LSCS]. FC = Fold-change -FDR = False Discovery Rate -LSCS = Low Somatic Cell Score line -HSCS = High Somatic Cell Score line

  The five most significantly enriched pathways in intercaruncular areas of Inoculated LSCS and Control LSCS ewes in Hallmark, KEGG pathways and Gene Ontology biological process terms gene sets. SIZE = Number of genes in the gene set after filtering out those genes not in the expression dataset. LSCS = Low Somatic Cell Score line -HSCS = High Somatic Cell Score lineDifferential Expression of [Inoculated LSCS] vs [Inoculated HSCS] in CAR areasBy comparing the expression profiles of the [Inoculated LSCS] to [Inoculated HSCS] ewes, 795 genes were found to be differentially expressed in CAR areas : 373 up-regulated in [Inoculated LSCS] ewes (264 FC>2) and 422 up-regulated in [Inoculated HSCS] ewes (321 FC>2) (Figure 4).

FIGURE 4 -

 4 FIGURE 4 -Volcano-plot and top 10 DEGs between [Inoculated LSCS] and [Inoculated HSCS] in CAR VOLCANO-PLOT : Orange dots are up-regulated DEGs in [Inoculated LSCS] ewes with absolute log2(FC)>2 and FDR<0.05. Green dots are up-regulated DEGs in [Inoculated HSCS] ewes with absolute log2(FC)>2 and FDR<0.05. The 10 shown DEGs are the 5 DEGs with the highest FC and the 5 DEGs with the lowest FC. TABLE : The 10 most significant DEGs between [Inoculated LSCS] and [Inoculated HSCS] in CAR. FC = Fold-change -FDR = False Discovery Rate -CAR = Caruncular areas -LSCS = Low Somatic Cell Score line -HSCS = High Somatic Cell Score line

FIGURE 5 -FC>2FIGURE 6 -

 56 FIGURE 5 -GO biological process categories of [Inoculated LSCS] vs [Inoculated HSCS] in CAR PIE CHARTS : Gene Ontology (GO) clusters from DAVID Functionnal Annotation Clustering were grouped according to their associated biological processes. Top chart was performed using DEGs up-regulated in [Inoculated LSCS]ewes and bottom chart using DEGs up-regulated in [Inoculated HSCS] ewes. TABLE :Top 10 enriched GO term clusters. Size = Number of DEGs involved in given term -% = Number of genes involved in given term divided by the total number of input genes -ES = Enrichment Score i.e. the geometric mean of all the enrichment P-values for each annotation term associated with the gene members in the group (Significative ES>1.3) -CAR = Caruncular areas.

FIGURE 7 -

 7 FIGURE 7 -GO biological process categories of [Inoculated LSCS] vs [Inoculated HSCS] in ICAR PIE CHARTS : Gene Ontology (GO) clusters from DAVID Functionnal Annotation Clustering were grouped according to their associated biological processes. Top chart was performed using DEGs up-regulated in [Inoculated LSCS]ewes and bottom chart using DEGs up-regulated in [Inoculated HSCS] ewes. TABLE : Top 10 enriched GO term clusters. Size = Number of DEGs involved in given term -% = Number of genes involved in given term divided by the total number of input genes -ES = Enrichment Score i.e. the geometric mean of all the enrichment P-values for each annotation term associated with the gene members in the group (Significative ES>1.3) -ICAR = Intercaruncular areas.

  Number of pathways up-regulated that reach significance with FDR<0.05 in [Inoculated LSCS] and [Inoculated HSCS] ewes. GSEA expresses all differences as upregulation to minimize class bias due to the intrinsic asymmetry of the method.CAR = Caruncular areas -ICAR = Intercaruncular areas -LSCS = Low Somatic Cell Score line -HSCS = High Somatic Cell Score line Indeed, [Inoculated HSCS] ewes display a large range of over-expressed immune pathways including acute phase response (IL6-JAK-STAT3 signaling), inflammatory processes (JAK-STAT signaling, Interferon alpha and gamma responses), innate immune response (Complement and Coagulation cascades), cytokine production or yet in adaptive immune response

  The five most significantly enriched pathways in caruncular and intercaruncular areas of Inoculated LSCS and Inoculated HSCS ewes in Hallmark, KEGG pathways and Gene Ontology biological process terms gene sets . SIZE = Number of genes in the gene set after filtering out those genes not in the expression dataset One of the most enriched KEGG pathway in [Inoculated HSCS] was Antigen Processing and Presentation. In this pathway, 15 genes were found significantly differentially expressed between [Inoculated LSCS] and [Inoculated HSCS] ewes : B2M, CALR, CANX, CD74, CD8B, CTSS, HLA-DOB, HLA-DRA, HSP90AB1, HSPA5, HSPA6, IFI30, LGMN, PDIA3 and PSME2 (Figure 8-B). GSEA analyses identified an enrichment of genes involved in the Primary Immunodeficiency KEGG pathway in [Inoculated HSCS]. In this pathway, 3 genes were found significantly differentially expressed between [Inoculated LSCS] and [Inoculated HSCS] ewes : CD40LG, CD8B and LCK (Figure 8-A).

FIGURE 8 -

 8 FIGURE 8 -Genes involved in Primary and Antigen Processing and Presentation A : Heatmaps generated by GSEA with genes involved in Primary Immundeficiency.B : Heatmaps generated by GSEA with genes involved in Antigen Processing and Presentation. Samples were labelled RU# which is an experimental identification. Encircled in red genes are significantly DEGs. Grey samples are [Inoculated LSCS] samples whereas orange samples are [Inoculated HSCS] samples. CAR = Caruncular areas -ICAR = Intercaruncular areas

FIGURE 9 -

 9 FIGURE 9 -Venn diagram showing CAR-specific, ICAR-specific and common DEGs from [Inoculated LSCS] vs [Inoculated HSCS] comparison Five CAR-specific, ICAR-specific and common DEGs were selected as exemples from DEGs with lowest FDR (False Discovery Rate = adjusted p-value). CAR = Caruncular areas -ICAR = Intercaruncular areas -LSCS = Low Somatic Cell Score line -HSCS = High Somatic Cell Score line

FIGURE 10 -

 10 FIGURE 10 -GO Biological process categories in DEGs for [Inoculated LSCS] vs [Inoculated HSCS] GO clusters from DAVID Functionnal Annotation Clustering were grouped according to their associated biological processes. Values indicated below biological process terms are the percentage of associated GO terms in all significantly enriched GO terms. CAR = Caruncular areas -ICAR = Intercaruncular areas

  This confirmation was required to justify the differential analysis by comparing [Inoculated LSCS] and [Inoculated HSCS] gene expression profiles. Expression levels of 795 genes in CAR areas and 2979 genes in ICAR areas were found significantly altered (FDR<0.05), attesting for a substantial difference between the two lines in term of endometrial response to a bacterial challenge. In both CAR and ICAR areas, up-regulated DEGs in [Inoculated HSCS] compared to [Inoculated LSCS] were involved in innate immunity, in cell-mediated and humoral adaptive immune responses as well as in inflammation and related pathways such as apoptosis or oxidative stress. On the opposite, up-regulated DEGs in [Inoculated LSCS] were mainly involved in cellular morphogenesis, tissue development, muscular and nervous processes. The enrichment of immunity GO terms for [Inoculated HSCS] ewes reflects that immune response is moving from innate immune response to humoral and cell-mediated adaptive immune responses over the 24h period. Interestingly, in the comparison between [Inoculated LSCS] and [Control LSCS], we did not find a significant number of DEGs involved in humoral adaptive immunity. Furthermore, the differential expression of immune genes between [Inoculated LSCS] and [Inoculated HSCS] ewes is of particular relevance, especially given observations from the morphometric analyses and the cytologic evaluation of uterine flushes. Indeed, almost 10 fold more neutrophils were found in the uterine lumen of HSCS ewes (1.26E+09 in total) than in LSCS ewes (1.39E+08 in total) whereas HSCS endometrium presented oedema formation and lower glandular density in ICAR areas (unpublished data, Foucras et al.).Increased cell migration and accumulation of fluid in the endometrium attest for a loss in a vascular permeability in the uteri of HSCS ewes. Moreover, in response to immune cell recruitment, endometrium of HSCS ewe underwent tissue distension in that could account for the under-representation of biological terms such as cellular morphogenesis, tissue development, muscular and nervous processes in this ovine line. These supplementary observations attest that uteri HSCS and LSCS ewes have a different morphological response to the local inflammation. Uterine flushes were also used to quantify the bacterial population in uterine lumens of ewes. We detected a significant difference between groups : 6.88E+07 in total in [Inoculated LSCS] ewes and 3.19E+07 in total in [Inoculated HSCS] ewes. Altogether, our findings suggest either an intensity and/or a time difference of immune response between lines in reply to the experimental infection, leading to a faster bacterial clearance of HSCS ewes.

SOCS2

  genotype show uncontrolled inflammation during infection despite pathogen clearance. Finally, the observation of oedema and endometrial tissue woundings leading to the formation of abcesses in uteri of [Inoculated HSCS] ewes (unpublished data, Foucras et al.) supports the hypothesis that a prolonged inflammation leads to tissue damages in HSCS line whereas LSCS line copes with metritis slowlier but with limited damaged of the endometrium.Prolonging and shortening the time of the experiment would be relevant to follow the evolution of inflammation, bacterial clearance, endometrial tissue recovery as well as endometrial function compatible with a pregnancy. Histological analyses are currently under investigation and will complete this study. Altogether, our data have shown that HSCS and LSCS ovine lines have a differential response to the infection regarding the immune cell recruitment, the inflammation of the endometrium and the activation of immune responses.

  comptage des Cellules Somatiques(CSC, SCS en anglais), du lait et qui présente donc une sensibilité divergente aux infections mammaires. La lignée [HSCS pour High SCS] est sensible aux mammites et la lignée [LSCS pour Low SCS] est résistante aux mammites. Cinq brebis [LSCS] et cinq brebis [HSCS] ont été expérimentalement infectées par voie intra-utérine avec une souche d'Escherichia coli spécifique de métrite (Sheldon, 2009), tandis que quatre brebis [LSCS] non infectées ont été conservées comme contrôles. Vingt-quatre heures après infection de l'endomètre, les utérus des brebis ont été prélevés et disséqués pour isoler les zones caronculaires aglandulaires (CAR) et intercaronculaires riches en glandes endométriales (ICAR). Les ARN totaux ont été extraits puis séquencés par RNA-seq. Les données de séquençage ont été traitées par FastQC, TopHat2 et featureCounts afin d'obtenir les profils d'expression des échantillons CAR et ICAR qui ont permis de réaliser les analyses différentielles avec DESeq2. L'analyse différentielle des échantillons ICAR entre [LSCS infectés] et [LSCS contrôles] révèle 199 gènes significativement différentiellement exprimés (DEGs, FDR <0,05). L'analyse fonctionnelle in silico a révélé que les DEGs sur-exprimés dans le groupe [LSCS infectés] sont majoritairement impliqués dans la réponse immunitaire innée, la réponse immunitaire acquise cellulaire et l'inflammation. Nombre de ces DEGS sont référencés dans la littérature comme marqueurs des métrites bovines (Kasimanickam et al.,2014), confirmant que la signature transcriptomique des infections utérines en ovin est similaire à celle publiée chez le bovin. Une seconde analyse différentielle a été réalisée entre les échantillons [LSCS infectés] et [HSCS infectés] pour les échantillons CAR et ICAR révélant 795 et 2979 DEGS respectivement. Les DEGS entre [LSCS infectés] et [HSCS infectés] ont été classé en trois groupes : CAR-spécifiques, ICAR-spécifiques et communs. Les DEGs communs sont majoritairement impliqués dans la réponse immunitaire innée et acquise et dans l'inflammation. Les DEGS CAR-spécifiques sont aussi impliqués dans la morphogenèse cellulaire et la signalisation intracellulaire et les DEGs ICAR-spécifique dans la morphogenèse cellulaire, le métabolisme des acides aminés et des lipides. La fonction biologique qui distingue les zones CAR et ICAR serait donc liée au métabolisme, ce qui s'expliquerait par l'activité 1 des glandes endométriales présentent dans les régions ICAR. Cette comparaison entre CAR et ICAR sera à approfondir uniquement avec une analyse différentielle prenant en compte l'appariement des tissus issus d'une même brebis. Les résultats de l'analyse différentielle ont mis en évidence un différentiel conséquent entre les lignées [LSCS] et [HSCS] en réponse à l'infection utérine expérimentale. L'analyse fonctionnelle in silico a révélé que les DEGs sur-exprimés dans le groupe [LSCS infectés] sont majoritairement impliqués dans la morphogenèse cellulaire et dans des processus nerveux et musculaires tandis que les DEGs sur-exprimés dans le groupe [HSCS infectés] apparaissent liés à l'inflammation à l'immunité innée l'immunité acquise à la fois cellulaire et humorale. Cet enrichissement en fonctions biologiques liées à la réponse immunitaire, associées à l'apparition d'une réponse humorale non observée entre [LSCS infectés] et [LSCS contrôles] et à l'observation d'une meilleure clairance bactérienne de l'utérus chez les animaux [HSCS], amènent à conclure que la lignée [HSCS] présente une réponse immunitaire à l'infection plus intense et/ou plus avancée que la lignée [LSCS]. Le développement d'une métrite suite à une infection peut avoir différentes causes : une réponse trop faible du système immunitaire ne permettant pas d'éliminer les pathogènes ou une inflammation locale excessive entrainant des lésions tissulaires de l'endomètre dans lesquels les pathogènes peuvent proliférer ou qui peuvent nuire à la fonction reproductrice de l'endomètre (Leblanc, 2014). Dans cette étude, nous avons donc relevé les deux hypothèses suivantes : (i) la lignée [LSCS] est plus résistante aux mammites mais plus sensible aux métrites que la lignée [HSCS], (ii) la lignée [HSCS] élimine mieux les pathogènes mais avec une inflammation excessive de l'endomètre pouvant faciliter de nouveaux épisodes de métrite. Notre analyse transcriptomique associée à des observations histologiques nous amènent à privilégier la seconde hypothèse. Tout d'abord, une voie KEGG d'immunodéficience primaire a été relevée comme significativement enrichie dans le groupe [HSCS] infectée. Ensuite, les brebis

TABLE 1 -Summary of GSEA analysis of [Inoculated LSCS] vs [Control LSCS]

 1

		Inoc. LSCS Control LSCS
	Hallmark	16	1
	KEGG	4	6
	GO biological process	83	10

Number of up-regulated pathways that reach significance with FDR<0.05 in [Inoculated LSCS] and [Control LSCS] ewes. GSEA expresses all differences as upregulation to minimize class bias due to the intrinsic asymmetry of the method. Hallmark = Hallmark gene sets are coherently expressed signatures derived by aggregating many Molecular Signatures DataBase (MSigDB) gene sets to represent well-defined biological states or processes. -KEGG = Gene sets derived from the KEGG pathway database -GO biological process = Gene sets derived from the GO Biological Process Ontology -LSCS = Low Somatic Cell Score line -HSCS = High Somatic Cell Score line

TABLE 2 -Top 5 of enriched gene sets from GSEA analysis Inoculated LSCS vs Control LSCS

 2

	Enriched in

TABLE 3 -GSEA analysis summary of [Inoculated LSCS] vs [Inoculated HSCS]

 3

		Inoc. LSCS	Inoc. HSCS
		CAR ICAR CAR ICAR
	Hallmark	2	3	18	22
	KEGG	1	0	30	34
	GO biological process	7	24	347	472

TABLE 4 -Top 5 of enriched gene sets from GSEA analysis of [Inoculated LSCS] vs [Inoculated HSCS]

 4

	Enriched in	CAR			ICAR
				Size FDR		Size FDR
		Inoc. LSCS	Myogenesis Spermatogenesis	157 95	1,38E-02 2,59E-02	KRAS signaling down Myogenesis Bile acid metabolism	148 157 100	1,35E-01 1,36E-01 2,02E-01
	Hallmark		Interferon gamma response	134	0,00E+00	Allograft rejection	149	0,00E+00
		Inoc. HSCS	Allograft rejection Interferon alpha response IL6 JAK STAT3 signaling	149 69 65	0,00E+00 0,00E+00 0,00E+00	Interferon gamma response MYC targets V1 MTORC1 signaling	134 146 159	0,00E+00 0,00E+00 0,00E+00
			Inflammatory response	154	0,00E+00	MYC targets V2	44	0,00E+00
		Inoc.	GAGs biosynthesis heparan sulfate	17	2,74E-02
		LSCS
	KEGG	Inoc. HSCS	Antigen processing and presentation Proteasome JAK STAT signaling pathway Cytokine cytokine receptor interaction	33 35 103 173	0,00E+00 0,00E+00 0,00E+00 0,00E+00	Proteasome Antigen processing and presentation Leishmania infection Intestinal immune network for IgA pro-	35 33 47 30	0,00E+00 0,00E+00 0,00E+00 0,00E+00
						duction
			Natural killer cell mediated cytotoxicity	74	0,00E+00	Hematopoietic cell lineage	56	0,00E+00
			Aminoglycan biosynthetic process	84	1,80E-02	Axoneme assembly	30	1,28E-04
		Inoc. LSCS	Regulation of cardiac conduction Regulation of neuronal synaptic plasti-	52 37	2,20E-02 2,50E-02	Cellular component assembly involved in morphogenesis Cilium movement	192 28	1,60E-04 2,13E-04
	GObp		city Proteoglycan biosynthetic process	48	2,70E-02	Cilium morphogenesis	158	3,19E-04
			Regulation of postsynaptic membrane	41	3,30E-02	Cilium organization	142	6,38E-04
			potential
			Adaptive immune response
		Inoc.
		HSCS

TABLE 5 -Selected genes for qRT-PCR

 5

	name	Alias	Function	FC CAR	FDR CAR	FC ICAR	FDR ICAR
	AGP	ORM1/2	Acute Phase Protein	0,11	2,27E-03	0,06	1,96E-05
	ALOX5AP	FLAP	Parturition and Inflammation	0,36	3,55E-02	0,31	3,91E-03
	C1R		Parturition and Inflammation	0,26	2,35E-06	0,28	3,97E-06
	C3		Parturition and Inflammation	0,18	2,75E-06	0,25	9,35E-05
	CCR2		Parturition and Inflammation	0,18	7,39E-07	0,14	7,64E-10
	CD14		Pathogens Recognition	0,43	1,24E-01	0,43	5,36E-02
	CLDN1		Uterine Tissue Remodeling	0,27	4,31E-04	0,48	4,54E-02
	CTSZ		Implantation	0,56	1,20E-01	0,26	3,64E-07
	CXCL12	SDF1	Implantation	0,48	9,62E-02	0,26	4,78E-05
	CXCR4	LAP-3	Implantation and Pathogens Recognition	1,07	9,22E-01	0,48	8,37E-03
	ESR1		Reproductive function	1,53	2,94E-01	2,37	1,50E-03
	FST		Reproductive function	1,31	6,72E-01	2,53	7,04E-03
	GPX3		Oxidative Stress and Antioxidant Defense	0,37	7,56E-02	0,22	8,07E-04
	H19		Genomic Imprinting	12,61	1,80E-04	4,91	1,83E-02
	IDO1		Implantation and Inflammation	0,22	2,05E-02	0,18	2,11E-03
	IDO2		Implantation and Inflammation	0,69	7,50E-01	0,05	6,03E-08
	IFNG		Cytokine	0,52	4,28E-01	0,18	5,55E-04
	IGF2		Genomic Imprinting	1,41	4,72E-01	2,37	4,10E-03
	IL10		Cytokine	0,48	3,51E-01	0,29	1,58E-02
	IL1B		Cytokine	1,48	7,65E-01	0,81	8,17E-01
	IL6		Cytokine	1,09	9,66E-01	0,86	8,84E-01
	IL8	CXCL8	Cytokine	2,53	3,58E-01	0,88	9,04E-01
	IL12RB1		Cytokine Receptor	0,41	7,75E-02	0,35	9,07E-03
	IL1R2		Cytokine Receptor	0,18	7,90E-03	0,16	1,23E-03
	LCN2	NGAL	Antibacterial protein	0,26	1,07E-01	0,11	5,76E-04
	LGALS9		Cytokine signaling	0,35	1,38E-02	0,29	7,33E-04
	LTF		Antibacterial protein	0,34	2,75E-01	0,18	1,25E-02
	MARCO	SCARA2	Antibacterial protein	0,59	6,68E-01	0,16	5,28E-03
	MD2	LY96	Pathogens Recognition	0,76	5,65E-01	0,51	1,80E-02
	MMP1	CLG	Tissue remodeling	0,15	1,29E-02	0,08	6,37E-05
	MSR1	SCARA1	Antibacterial protein	0,25	1,42E-06	0,34	1,25E-04
	MSX1	HOX7	Uterine Tissue Remodeling	2,84	8,05E-02	3,38	9,91E-03
	MUC13		Antibacterial protein	0,17	1,81E-02	0,12	9,92E-04
	PLAC8		Implantation	0,18	4,05E-04	0,24	1,83E-03
	PLXNC1	VESPR	Acute Inflammatory Response	0,35	1,52E-09	0,30	6,15E-14
	S100A8		Antibacterial protein	0,20	4,68E-02	0,10	3,48E-04
	SAA4		Acute Phase Protein	0,26	4,10E-02	0,18	1,71E-03
	SOD2		Oxidative Stress and Antioxidant Defense	0,53	4,29E-01	0,37	6,68E-02
	TIMP1		Tissue remodeling	0,56	4,08E-01	0,21	4,08E-04
	TNFA		Cytokine	1,35	7,87E-01	0,96	9,68E-01
	TLR8		Pathogens Recognition	0,33	1,58E-03	0,21	5,01E-07
	WARS	TrpRS	Implantation	0,23	1,39E-02	0,24	5,47E-03

Grey table cells are

FDR<0.05. FC = Fold-Change [Inoculated LSCS]/[Inoculated HSCS] -FDR = False Discovery Rate -CAR = Caruncular areas -ICAR = Intercaruncular areas

Remerciements


Virginie Svenningsen
email: virginie.svenningsen@mines-paristech.fr

Sub-theme 32: Hybrid Organizations and Organizing: Coping with Uncertainty and Creating the Unexpected

Keywords: Hybrid Organizations, Institutional Logics, Institutional Complexity, Business Models

In a context of new academic-industrial collaborative research organizations, this case study explores the use institutional logics in developing a portfolio business model able to keep stakeholders' engagement. The co-existence of dual dominant logics in developing the business model associated with knowledge value creation has two consequences. It allows maintaining stakeholders' engagement, whereas the hybrid organization combining the academic and commercial logics will loose actors' motivation. It also facilitates actors' engagement in a new logic and the development of the two other business models aimed at creating societal and economic value. This research contributes to understanding how the strategic use of logics can help new hybrids developing and sustaining. It also offers a relative new lens of analysis in the institutional literature: the business modeling.

INTRODUCTION

Organizations have come to realize that joining resources can be a successful strategy for addressing societal and environmental issues [START_REF] Haigh | Hybrid organizations: The Next Chapter of Sustainable Business[END_REF]. While different definitions of hybrid organizations are being proposed, all of them recognize that hybrid organizations respond to the inability of one of the partners to solve an important problem [START_REF] Borys | Hybrid Arrangements as Strategic Alliances: Theoretical Issues in Organizational Combinations[END_REF], such environmental issues, by enhancing collaborative R&D efforts in sustainable energies. However, new hybrids organization in this sector cannot rely on existing models, in how creating value for multiple stakeholders and R&D activities take time deliver values and convince stakeholders to maintain their engagement! This research analyzes how new hybrids in this field, used differently and progressively, institutional logics in their business modeling. This case is of particular interest, as they were based on the same state bid offer, but developed differently: one stopped and the other experienced a crisis but managed to overcome it.

CONCEPTUAL BACKGROUND

Hybrid organizations the interface between for-profit and nonprofit sectors, have a potential advantage to merge together distinctive capabilities and different resources.

They fast developed, as they can address social and ecological issues [START_REF] Haigh | The New Heretics: Hybrid Organizations and the Challenges They Present to Corporate Sustainability[END_REF]). But, in order to do that, they also have to face the complex problems of institutional complexity that occur when parties from different organizational fields, characterized by different logics collaborate. Literature refer to institutional complexity when organizations "confront incompatible prescriptions from multiple institutional logics" [START_REF] Greenwood | Institutional complexity and organizational responses[END_REF].

By adopting structures and practices regarded as legitimate by wider audiences, organizations received social approval and access to critical resources [START_REF] Raynard | Deconstructing complexity: how organizations cope with multiple institutional logics Academy of Management Proceeding[END_REF]. By adopting structures and practices from multiple logics, hybrid organization span social categories and take the risk of confusing their audience and suffer legitimacy discount [START_REF] Rao | Border Crossing-Bricolage and the Erosion of Categorical Boundaries in French Gastronomy[END_REF]. At the intra-organizational level, the convergence of multiple logics makes organizations vulnerable to incoherence because advocates of different logics compete to impose their normative beliefs and interest [START_REF] Battilana | Advancing Research on Hybrid Organizing[END_REF]. Hybrids can also face multiple internal challenges, in terms of member identification [START_REF] Glynn | When Cymbals Become Symbols-Conflict Over Organizational Identity Within a Symphony Orchestra[END_REF], internal conflicts [START_REF] Ashforth | Functions of Dysfunction: Managing the Dynamics of of an Organizational Duality in a Natural Food Cooperative[END_REF] and cognitive tensions [START_REF] Foreman | Members' Identification with multiple-identity Organizations[END_REF]. New hybrids face also the challenge of maintaining hybridity, as they develop. Intraorganizational power struggles or shifts in the influence and engagement can influence balance commercial and societal elements, phenomenon referred to as "mission drift" [START_REF] Ebrahim | The governance of social enterprises: Mission drift and accountability challenges in hybrid organizations[END_REF][START_REF] Santos | Making Hybrids Work: Aligning Business Models and Organizational Design for Social Enterprises[END_REF].

Despite those challenges, hybrids may be able to benefit from untapped resources.

They may for example broaden their potential resource by including minority logics appealing to new audience [START_REF] Durand | Jules or Jim: Alternative Conformity to Minority Logics[END_REF]. They may antagonist assets into opportunities [START_REF] Hockerts | How Hybrid Organizations Turn Antagonistic Assets into Complementarities[END_REF]. Hybrids heterogeneity can also offers opportunities for innovation [START_REF] Dalpiaz | Combining Logics to Transform Organizational Agency: Blending Industry and Art at Alessi[END_REF] [START_REF] Jay | Navigating Paradox as a Mechanism of Change and Innovation in Hybrid Organizations[END_REF]. Such cases prove that organizations can mitigate risks associated with institutional complexity and leverage from institutional plurality [START_REF] Kraatz | Organizatiobal Implications of Institutional Pluralism, The Sage Handbook of Organizational Institutionalism[END_REF], 2017).

Different organizational strategies have been suggested, in order to cope with institutional pressures [START_REF] Oliver | Strategic Responses to Institutional Processes Academy[END_REF]. As hybrids must response to expectations from multiple institutional spheres, strategies consider specific dimensions, such tensions related to multiple logics on goals or means [START_REF] Pache | When Worlds Collide: the Internal Dynamics of Organizational Responses to Conflicting Institutional Demands[END_REF]. The relative level of compatibility and centrality between logics can also influence which strategy to adopt (Besharov & Smith, 2013) (p.365).

The analysis of new bank formation, highlights how a commercial and community logics both influenced the process of establishing a bank [START_REF] Almandoz | Arriving at the Starting Line: The Impact of Community and Financial Logics on New Banking Ventures[END_REF]. This research invites investigations into how and under what conditions different logics can assist or hinder new hybrids performance and outcome.

Business model can be used as a pivotal level and unit of analysis in our understanding of value creation and capture [START_REF] Zott | The business model: A theoretically anchored robust construct for strategic analysis[END_REF]. A portfolio of business models can be defined as "the range of different ways a firm delivers value to its customers to ensure both its medium term viability and future development" [START_REF] Sabatier | From Recipe to Dinner: Business Model Portfolios in the European Biopharmaceutical Industry[END_REF]. A business modeling process approach suggests that learning, signaling, convincing through business modeling process facilitate legitimation process [START_REF] Bojovic | Learning, signaling, and convincing: The role of experimentation in the business modeling process[END_REF], which can be very relevant in the case of new hybrid formation. Literature indicates that hybrid organization foster heterogeneous and innovative BM [START_REF] Ocasio | Strategy and commitments to institutional logics: Organizational heterogeneity in business models and governance[END_REF], and interesting and effective business model innovations [START_REF] Santos | Making Hybrids Work: Aligning Business Models and Organizational Design for Social Enterprises[END_REF].

But they say little about how this is done and with which influence of stakeholders' engagement in new hybrids.

This introduces my research question:

How can the use of institutional logics in developing the business model of new hybrids, influence the level of multiple stakeholders' engagement?

EMPIRICAL SETTING AND METHOD

The institutional field of French collaborative R&D includes various types of private/public, public/public or private/private partnerships. Three institutional logics characterize this field: professional, commercial and state logics.

-------------INSERT TABLE 1 ABOUT HERE-------------A large initiative (PIA 1 ) is launched by the French state in 2011, in order to fund up to 50% of new independent legal entities. Our research focuses on four of the thirteen REI 2 , created as a result from the PIA. The four organizations analyzed were all based on the same PIA bid-offer conditions and they were all created legally in 2013. For confidential reasons, we call them Solar Energy, Industrial Efficiency, City Efficiency and Ocean Energy. Governance structures must enable a balance between industrials, SME and academics actors, for jointly co-design and co-run the REI in order to create multiple types of values. For simplification purpose, we call E, the economic value; K, the knowledge value creation and S, the societal value proposition. Given this requirement, we use a business model lens for analyzing how organizations map resource to value creation and how they use them logics as strategic resources.

The four REI have different TRL3 levels, which can influence their business modeling.

An overview of the four REI can be found in the table below:

- ------------INSERT TABLE 2 ABOUT HERE-------------We adopt a case study method [START_REF] Yin | Case Study Research: design and methods[END_REF], in order to compare patterns and distinct elements explaining our research question. This research is based on primary data (30 semi-structured interviews, all transcribed and coded, annual reports, meeting notes, state reports…) and secondary data (third-party analysis).

FINDINGS

1. First findings: first BM (K) design using dual logics co-existence or combination influence the level of stakeholders' engagement:

• Dual logics' related to multiple goals influence stakeholders collective engagement, whether they collectively selected and co-exist or whether they were not collectively decided and are combined:

"There is also one point that is very important, and which varies according to the type of hybrid organization, is the way the projects are built. At Solar Energy, projects were, were co-built between industry and the academic world has allowed a whole range of graduations in the positioning of projects between things that are fairly applied and then things that remain very upstream, which are exploratory, with TRL both very low and then intermediate TRLs. It offers a portfolio of projects with a large variety of technologies." (Solar Energy academic partner MA)

Other quotes below: -------------INSERT TABLE 3 ABOUT HERE-------------

• Dual logics' related to multiple means influence stakeholders collective engagement, whether they co-exist or whether they are combined:

➢

Taking into account the need to respect the multiple norms and practices into account:

"On one of the projects, the project manager is currently an industrialist but we are changing this currently into the hands of an academic. Why? Well, it turns out, this is finally a rather exploratory subject, where we will need more time, science, understanding of the mechanisms applied and therefore, we are changing the project manager; there is no standardized process on who shall rather be project manager, it depends on competences and need." (Solar Energy project coordinator)

Other quotes below:

-

------------INSERT TABLE 4 ABOUT HERE-------------

➢

Leveraging from the differences in norms and practices and facilitate coexistence:

"The management of the collaborative institute must give the structure, the rules of the game but inside it must leave room ... inside, let the academic and industrial partners collaboratively decide which methods to prefer according to the subjects ...

Otherwise, everyone does not meet their interests and the institute finally becomes a threat when at the base, we had worked to make it an opportunity. " (Energy Efficiency academic partner)

Other quotes below:

- Other quotes below:

------------INSERT TABLE 5 ABOUT HERE------------- ➢ Collective
-

------------INSERT TABLE 6 ABOUT HERE------------- 2.
Second findings: influence of the first BM design on stakeholders' motivation to engage further developing the two other BM with an additional minority logic:

• Leveraging on design of the first BM with dual logics co-existence, facilitate stakeholders engagement in developing the other portfolio BM, including new logics goals and means, that may not be as dominant:

"The ground idea is that Solar Energy is able to perform collaborative research, and that I think it meets the needs of the state and our partners. Now, the commercial side of Solar Energy, is a project we launch this year. How, is it possible for mid-sized companies and other partners to use the Solar Energy platform, which is essential because according to our business plan, intellectual property contributes 8%, but I think it will never happen. Our business plan is not based on the value of research, but on the valuation of its platform, its building and its equipment" (…) We must also continue to develop objectives that are not necessarily linked to research programs.

For example, developing partnerships in France, internationally, finding new resources, developing services, but also subsidies, being able to lobby the European Community, etc. " (Solar Energy management).

Other quotes below:

- Other quotes below:

------------INSERT TABLE 7 ABOUT HERE-------------
-

------------INSERT TABLE 8 ABOUT HERE-------------

DISCUSSION AND CONCLUSION

As in the Drug Court case, whereby professionals used multiple logics pro-actively, logics resemble tools, creatively employed by actors to reach their goals of the value creation, they are most interested in [START_REF] Mcpherson | Logics in Action: Managing Institutional Complexity in a Drug Court[END_REF]. This research also suggests that use of logics is only supporting stakeholders' engagement when it is coexisting and not when it is combined. This is due to the fact, that the practices enabling logics co-existence increase collective learning, mutual understanding, which tends to decrease in practice incompatibility between logics. This study also suggests that, institutional logics can indeed represent strategic resources organizations can use to leverage their strategic choices [START_REF] Durand | Institutional Logics as Strategic Resources[END_REF]. However this is more complicated in the case of hybrid where the management and the governance includes managers embedded in multiple logics and with different norms and interests in using strategically the available logics.

Analyzing how new hybrid organizations develop portfolio management using logics, allows us to see the effect of a first business model strategic choice for the hybrid capacity to develop the two other business models associated with other goals and means, using both the benefits associated with formalization and collaboration, [START_REF] Ramus | Institutional complexity in turbulent times-formalization, collaboration, and the emergence of blended logics[END_REF] through the business modeling. Such process also enhance stakeholders' engagement, as it fosters learning, signaling and convincing [START_REF] Bojovic | Learning, signaling, and convincing: The role of experimentation in the business modeling process[END_REF] among the multiple stakeholders. Adopting a more dynamic analysis of new hybrid organizing strategies also meet the call for further analysis on this perspective [START_REF] Battilana | On hybrids and hybrid organizing: a review and roadmap for future research[END_REF]. -"I consider that Solar Energy a facilitator of construction of collective projects between the industrialists of the sector and the academic researchers. This organization is in no way prescribing it as a structure (...) The management is more manager of this structure, more than the prescriber, in the negotiations between industrial and academic actors to define projects " (Solar Energy academic partner MA)

-"At Solar Energy, we're not in collaborative research led by intermediary groups or order givers, but in a joint search. Organizations that mediate relations between industrial and academic, that's just no, we can work very well with them." (Solar Energy academic partner LE) -"Most of the time, the heads of the twelve projects are with the partners because we at Solar Energy do not necessarily have the competence or even the wish to coordinate. The project is under the responsibility of the project manager, a single project manager, sometimes academic, or industrial, or Solar Energy, it depends.

To decide, we look in the Solar Energy community who is best able to coordinate this project. Sometimes they are academics, sometimes depending on the subjects, the projects with a low TRL, rather basic, at 80% are academic project leaders.

The projects with high TRL, where we are closer to the pilot line, will be more at the industrial (high it will be from 5 and low it is 3.)" (Solar Energy Project Coordinator)

-"The coordination between the projects and the various partners, is done through the scientific director (50% of his time in academic lab) and myself, industrial researcher, but made available at Solar Energy for three years. So I am more operational and the scientific director allows the scientific balance and is more strategic." (Solar Energy Project Coordinator)

-"Another important interest for the academic world, and in my opinion also important for the industrialists, is to share the target. This is not so obvious that the target in terms of performance, in terms of cost, in terms of the machine, it is to have a roadmap co-built there also between industrialists. For the academic world, it is also very important, to have this visibility there it allows to make choices. we discover something, but we realize that we have all the evidence but that we will not arrive in fine with the objective it will be able to be abandoned, even if it is interesting in itself". (Solar Energy academic partner MA)

-"We have a certain number of research units that are doing research, albeit relatively upstream, but with applicative aims, and rubbing shoulders with reality, well it works out pretty well." But to succeed in the industrial stage and to keep the same properties as what we saw in the lab, it's not quite the same: we value a lot better when working with industrialists ". (Solar Energy academic partner MA)

HR norms: "This form of hybrid organization takes us completely out of our preferred mode of collaboration with industrialists, which is a way of participating in collaborative project projects. (…) The availability is at the researcher's choice, it's his status; up to him to decide whether it's going to be available or not. Therefore, in the construction of these hybrid organizations, it can be a problem, because we cannot operate according to private sector norms! At Solar Energy, they accepted this and therefore our researchers work collaboratively, without contracts with them, they participate in projects, but without being made available, without reporting to the formal departments of Solar Energy." (Solar Energy academic partner MA)

Reporting norms: "Industrial research is still very difficult for academics One of our three program managers (academic partner) does not want to continue because he says: I spend too much time in the administration, in reporting, I want to bring ideas, collective intelligence" (Industry Efficiency Management).

TABLE 5: TABLE 6:

Academics norms: "If we succeed in making sure that researchers feel respected in the sense that they must do basic research, that they communicate ... But on the other hand, they are embedded in applied projects, applications, with a industrial sense, that, for the academics, it would be a real success to get to do it, and so they find there in our institute, an experimental object for them, to be able to combine academic research and applicability of their research." (Solar Energy management)

Intellectual Property practices: "The industrial partners derive a primary benefit from the results that come out of the research ... But beyond that, we are really on a principle of co-ownership, compared to the shares of intellectual property (...). There are actually the interests of the partners at first, but for example at Solar Energy, they want to ensure co-ownership to ensure the sustainability of the organization (...), it's really a partnership work ... " (Solar Energy academic partner MA)

-"I think it's better not to singularize the objectives according to industrialists, academics ... We could give ourselves roles in the institute, say:" The industrialists do that, academics do that, but no, not more It does not exist, we have not thought about it, we just take the partners, their specificities: the academics with their skills that do not necessarily have a great idea of what a project can be, and industrialists with a vision that is still much more industrial, finality, value. The idea of our institute, our added value, is to rely on these differences, to take the best of these differences to do things that is new and different from what was done before, so yes, maybe ask the industry to set the long-term objectives in technological terms, to say, "We need 30% yield in 2030," and for get there to rely on academic skills that will tell us, to have ideas on how can we do it, what science can be applied... But, after that, we need joint projects between the different types of researchers , without saying "your difference is this one and rather than that", while you want to create projects where everyone works together. The idea is to try to erase these differences rather than to stigmatize them, knowing that these differences exist" (Solar Energy management)

TABLE 7:

-"At the very beginning of the institute, we were more on a push R & D logic where the science pushed subjects and was concerned only after knowing how it will happen in the market.I think there is an evolution that Today and even if the scientific director comes from the academic and is much more push, they reach, through the questions that we address, and by the project leaders are academic, but also industrial to appropriate objectives more related to the constraints of the economic environment " (Solar Energy industrial partner)

-"In each project team, there are at the same time researchers Solar Energy, academic and also industrial partners". (Solar Energy project coordinator)

-"The feedback we have, including at the level of the scientific actors, is that the projects co-joined between academic researchers, industrialists allows them to learn to work together, despite different communities, histories, objectives different, relying on people who all ultimately have a technical sense, it has really come into existence, there is indeed trust and teamwork that is being put in place." (Solar Energy industrial partners)

-"Nobody was in a logic: I take 5 researchers from my home, 5 researchers from home, we put all these researchers together ... In this case, academic researchers do not care, because then works according to its own model and its own way of doing things and we do not care about the collaborative institute." (Industry Efficiency academic partner)

-"Industry Efficiency would have had to rely on each other's skills and combine them, so that people can help each other (...) For example, to help the partner who has the skills I need It was the problem of Zodiac and Airbus, for example, and finally Airbus financed Zodiac so that they could give them the seats. But Industry Efficiency did not organize that ... " (Industry Efficiency academic partner)

-"The leaders of the institute announce: we will do energy audits. I said: ok, we need to be there. They answer: Oh no, no, you at the research laboratory, your role is when there's a research topic, we call you in. (…)" I said it does not work, we want to be in direct contact with companies to be immersed in the issues, but no, that was not their way!" (Industry Efficiency Academic Partner)

-"These institutes need to bring together public and industrial interests to create assets that enable the development of new products and services to improve the competitiveness of some and meet the public objective of making widely available alternative solutions to the carbon economy. " (State actor)

-"One of the original features of this type of hybrid organization and their main potential advantage, and this is where they should focus their research, it is the pooling of long-term issues. If two industrialists get together, they do not necessarily have the same business interests, but they can at least share a vision to long term, and also share it with the smallest companies or another company that will also have different interests. But together, it makes several manufacturers in the same industry that get together to define common projects, and that, this is not usual but very interesting to be involved in (...)" (Solar Energy academic partner).

-"On the other hand, very little has been done from our side, regarding the economic model, as this is not our business, .the way they choose to build the economic equilibrium eventually ..." (Solar Energy academic partner)

-"To transform the critical mass, we have to see that the institute exists and that it brings out results that count in the entire photovoltaic community. To do this, we need recognition of the brand" Solar Energy " externally, through communications, it is this scientific recognition, which will make it possible to valorize a certain number of results of the IPVF, for example that big industrialists can be in dialogue with us to buy patents. is also the one that will allow the institute to attract international researchers, that Japanese, German or American researchers want to spend a year at Solar Energy, with the equipment and all types of researchers in this field, linked to the same "Solar Energy" banner, when we have succeeded in attracting researchers of the highest level, this will facilitate our non-state funding in 2014, and will ultimately help us t succeed in our business model business and ensure our sustainability ". (Solar Energy industrial partner)

-"Solar Energy is also a question of industrial policy: will there be a European PV industry or not? Or at least on process bricks, it is this reality of an industrialization of significant photovoltaic components in Europe that is at stake or at least on process bricks, it is this reality of an industrialization of significant components of photovoltaïcs in Europe. It is also succeeding in connecting small and medium-sized companies, large groups in order to create jobs. Succeeding in this, could help determine how we are able in Europe to rely on the reality of an economic sector, generated in Europe through collaborative institutes like Solar Energy, to create a virtuous loop. " (Solar Energy industrial partner)

-" We must now respond to a broader objective, we will say of general public interest, despite the fact that it is also very concrete. The energy transition is what we develop and the creation of values we are expecting, must serve beyond the consortium, of our 13 private shareholders. We need to develop tools, methods or a specific skill that we will put at the service of the actors of the city, who need them, Beyond our shareholders, our shareholders, our 13, represent almost all the major categories of urban actors who are confronted with the energy transition." (City Energy management)

-"We have received funding from the state since 2015, but from 2012 to 2015, we worked exclusively on the basis of funding from our members regions, so six regions, which is very different from the usual setting of these institutions REI ... (...) In our initial project, there was the juxtaposition of an activity of tests and validations at sea with R & D programs, and there is a whole part of the project of the labeled project which finally is not retained by the state. So, first we had to change our objectives at the end of 2014, by eliminating two of the five test sites that were initially proposed, finally the state made us understand that it wanted to finance the test sites only independently (...) I think there is really a question of lack of confidence in the sector's capacity We have therefore both changed techno choices, but also a financing structure with the state, based on project and not directly to our legal entity (…). Beside the changes in techno objectives, and in the long term we are now relying more on objectives linked to societal rationales linked to the development of renewable and also economic energies -that is to say, that there is at the same time working on the relevance of marine renewable energies in the energy mix to be developed in the context of the energy transition and the development of an economic sector in what is called the economy 2 and thus allowing the conversion of shipyards and their workforce. It implies developing skills, systems, etc., that can be exported (...). So, there is clearly a vision that is to develop a French economic sector for export. That's why we consider even more then importance of looking at what's happening overseas, because we're coming into really different paradigms, a cost of much higher energy produced, which makes marine energy relevant much earlier than in metropolitan France. This is an excellent showcase, because here, in the French overseas, we can afford experiments that can not allow other places not connected to the network of archipelago countries, islands, etc., and so it is better to advance on overseas, and it opens these markets. " (Ocean Energy management)

-"The fact that we conduct research in consortium with most of the French actors allows us to formulate opinions that we share with the members and then that we propose in these international circles whether it concerns the actual research, the standardization or the certification, and it is really important to have a French voice in bodies that are, in the case of EMR, very strongly Anglo-Saxon. So, despite the lack of central state support, our industrial members and regions, fully understand the interest of that and they are willing to finance it." (Ocean Energy management)

•

  Not being able to leverage from the first BM limits stakeholders' motivation to engage further in new portfolio BM development or force changes in BM goals and means, in order to maintain stakeholders left engaged:

	"We had to do a training, which we did besides which was the professional training
	on the energy audit (...). We wanted to create a collective intelligence on training and
	it was very disappointing in many things. The academics did not know how to
	integrate Industrial Efficiency in their reflection (...). They were quickly withdrawn,
	they already had their work to do compared to the university and the Master. The
	industrialists who said "yes, we are very interested in being trained on energy
	efficiency" have finally been very behind the scenes too. They were posting this, but it
	was not a real priority". (Industrial Efficiency manager)

TABLE 1 TABLE 2 :

 12

	Characteristics	Academic logic	Commercial Logic	State Logic
	Economic system	Not-for profit funding	For profit funding	Public/not-for-profit funding
	Organizational Identity Science as profession	Science as business	Science as societal well-being
	Basis for legitimacy	Scientific reputation & legitimacy	Succesful Innovation	Societal improvements
	Authority structures	Craft-based authority Scientific autonomy	Accountability to business Leaders	Accountability to public authorities
				Use knowledge for
	Mission	Pursue scientific novelty	Use knowledge to develop new products	societal missions (environment, training,
				employement)
	Strategy	Attract research funding Long term creation of knowledge Publishing practices	Research undertaken only if change of profitable exploitation	Incentives to attract Industrial funding & Academic knowledge
	Value Proposition	Increase scientific knowledge	Economic return from scientific research activities	Create educational training Improve Energy transition
	Ideal Types adapted from Thornton (2002)

TABLE 3 :

 3

				Names of Collaborative Research Legal Entities *
	Date	Description	Solar Energy	Industrial Efficiency	City Efficiency	Ocean Energy
		Creation of legal entity Private form	Public form	Private form	Public form
	2013	Central French state labelized? Central French state funding?	Yes Yes, through legal entity Yes, through legal entity Yes (after 2nd attempt)	Yes Yes, through legal entity	No through legal entity Yes but by project, not
		Business model (BM) portfolio plan	E + K + S **	K + S **	E + K + S **	E + K + S **
	2013-2016	BM Implementation status	K first, S second K, S progressively interconnected	K only S attempted, not succesful	K first, S second, E third K and S simultaneously interconnected interconnected K, S, E progressively K and S progressively
			-Yes, ongoing	-Yes, ongoing	-Yes, ongoing	-Yes, starts
	2015	Central French state funding?	-Funding through legal entity	-Funding through legal entity	-Funding through legal entity	-Funding through distinct projects (not to
						the legal entity)
	end 2016	BM objectives vs planned in 2013	E + K + S Unchanged in the goals Changed in the goals (K only) Unchanged in the goals Unchanged in the goals K E + K + S E + K + S
			Unchanged in the means Changed in the means	Changed in the means	Changed in the means
	2017	BM Implementation	K & S operational E in development	Legal entities disolved	K & S operational E in development	K & S operational E in pre-development
		status	Interconnection between		Interconnection between	Interconnection
			K, S, E		K, S, E	between K, S, E
	* Not realy names for confidentially purposes	** K: Knowledge Value Proposition for the legal entity and shareholders
	** E: Economic Value proposition for the legal entity	** S: Societal Value Proposition for Stakeholders and society

TABLE 4 :

 4 -"In these hybrid organizations, the collaborative work goes pretty well provided that the project was actually co-built with us academically (…) At Solar Energy, it's relatively Well, a Solar Energy project manager is not going to say to a university professor, "Well, that's what we have to do now, and here is how..." It does not work that way, they'll talk... " (Solar Energy academic partner MA)

PIA: Plan d'Investissement d'Avenir (Future Investment Plan)

Renewable Energy Institutes (Instituts de Transition Energétique)

TRL: Technological Readiness Level