MD Jean-Laurent Casanova
email: jean-laurent.casanova@rockefeller.edu

Jacinta Bustamante

Andres Augusto Arias

Guillaume Vogt

Capucine Picard

Lizbeth Blancas Galicia

Carolina Prando

Audrey V Grant

Christophe C Marchal

Marjorie Hubeau

Ariane Chapgier

Ludovic De Beaucoudrey

Anne Puel

Jacqueline Feinberg

Ethan Valinetz

Lucile Jannière

Céline Besse

Anne Boland

Jean-Marie Brisseau

Stéphane Blanche

Olivier Lortholary

Claire Fieschi

Jean-François Emile

Stéphanie Boisson-Dupuis

Saleh Al-Muhsen

Bruce Woda

Peter E Newburger

Antonio Condino-Neto

Mary C Dinauer

Laurent Abel

Germline but macrophage-tropic CYBB mutations in kindreds with X-linked predisposition to tuberculous mycobacterial diseases

Keywords: tuberculosis, BCG vaccine, macrophages, NADPH activity

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Tuberculosis is a leading public health problem worldwide and the study of genetic predisposition to tuberculosis is a promising avenue of research [START_REF] Casanova | Genetic dissection of immunity to mycobacteria: the human model[END_REF][START_REF] Alcais | Tuberculosis in children and adults: two distinct genetic diseases[END_REF] . Mendelian susceptibility to mycobacterial disease (MSMD; MIM 209950) is a rare syndrome, resulting in predisposition to clinical disease caused by weakly virulent mycobacterial species, such as tuberculous Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccines and non-tuberculous, environmental mycobacteria [START_REF] Casanova | Genetic dissection of immunity to mycobacteria: the human model[END_REF][START_REF]Inborn errors of IL-12/23-and IFN-gamma-mediated immunity: molecular, cellular, and clinical features[END_REF] . These patients are also vulnerable to more virulent M. tuberculosis [START_REF] Alcais | Tuberculosis in children and adults: two distinct genetic diseases[END_REF] . Five disease-causing autosomal genes (IFNGR1, IFNGR2, STAT1, IL12RB1 and IL12B) and one X-linked gene (NEMO) have been found [START_REF]Inborn errors of IL-12/23-and IFN-gamma-mediated immunity: molecular, cellular, and clinical features[END_REF] . Allelic heterogeneity accounts for the existence of 13 distinct disorders, all of which impair IFN-γ-mediated immunity. The genetic etiology of about half of the patients with MSMD however remains unclear. We previously reported four maternally related French male patients (kindred A, P1 to P4) presenting a second X-linked recessive form of MSMD [START_REF] Bustamante | A novel X-linked recessive form of Mendelian susceptibility to mycobaterial disease[END_REF] (XR-MSMD-2) (Fig. 1a). The patients had recurrent or disseminated tuberculous mycobacterial disease: BCG-disease in three patients (MSMD sensu stricto) and tuberculosis in one (not vaccinated by BCG). We recently identified another French kindred with XR-MSMD; the three male patients of this kindred (kindred B, P5 to P7) had BCGdisease. In patients from both kindreds, there was no distinguishable immunological phenotype and the known etiologies of MSMD, including, in particular, mutations in X-linked NEMO [START_REF]X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production[END_REF] , were excluded, suggesting that the two kindreds may share a new XR genetic etiology of MSMD.

Results

CYBB mutations associated with MSMD

Multipoint linkage analysis in the two kindreds (Methods and Supplementary information) gave a maximal LOD score of 2.29 for two candidate regions on Xp11.3-Xp21.1 (13.83 Mb) and Xq25-Xq26.3 (11.79 Mb) (Supplementary Fig. 1a). Known X-linked primary immunodeficiencies (PIDs) were previously excluded on clinical and immunological grounds in both kindreds. We nonetheless sequenced the coding region of the PID-causing genes present in these two chromosomal intervals, using DNA isolated from the two probands (P4 in kindred A and P5 in kindred B). No mutations were found in TNFSF5, encoding CD40L (CD154) [START_REF] Notarangelo | Primary immunodeficiencies: 2009 update[END_REF] but a nucleotide substitution (A->C) in the codon for amino acid position 231, resulting in the replacement of a glutamine by a proline residue (Q231P), was found in exon 7 of CYBB in P4 from kindred A (Fig. 1a andb). A nucleotide substitution (A->C) in the codon for amino acid position 178, resulting in the replacement of a threonine by a proline residue (T178P), was found in exon 6 of CYBB in P5 from kindred B (Fig. 1a andb). Mutations of CYBB are commonly associated with chronic granulomatous disease (CGD) [START_REF] Roos | Hematologically important mutations: X-linked chronic granulomatous disease (third update)[END_REF] . In both kindreds, the clinically affected male subjects were all hemizygous for the mutant allele, whereas the other maternally related, healthy male subjects tested were not. The eleven obligate female carriers tested in the two kindreds were heterozygous, including an 90-year-old woman with a history of severe tuberculosis (H1), as well as four other female subjects (Fig. 1a). The strict familial cosegregation of the CYBB genotype and the MSMD phenotype (extended to tuberculosis) in the male subjects alive from both kindreds suggested that the CYBB mutations were responsible for disease. The mutations must have been transmitted by the male founders of both kindreds in generation I, although they did not display MSMD (Fig. 1a). These men however lived in France before the introduction of routine BCG vaccination in children, at a time at which tuberculosis was already declining. The Q231P and T178P CYBB alleles were not found in any of 1,300 X chromosomes from 52 ethnic groups (in the HGDP-CEPH panel), including 240 European chromosomes. Moreover, the two mutations are non-conservative, both substituting a Proline which is particularly disruptive (Fig. 1c), and affect residues that are conserved in the 33 animal species studied (Supplementary Fig. 1b). Finally, these two mutations have not previously been associated with CGD, a well known PID which is associated with multiple bacterial and fungal infectious diseases, including tuberculous mycobacterial diseases [START_REF] Roos | Hematologically important mutations: X-linked chronic granulomatous disease (third update)[END_REF][START_REF] Mouy | Incidence, severity, and prevention of infections in chronic granulomatous disease[END_REF][START_REF] Segal | Genetic, biochemical, and clinical features of chronic granulomatous disease[END_REF][START_REF] Winkelstein | Chronic granulomatous disease. Report on a national registry of 368 patients[END_REF][START_REF] Bustamante | BCG-osis and tuberculosis in a child with chronic granulomatous disease[END_REF][START_REF] Lee | Susceptibility to mycobacterial infections in children with X-linked chronic granulomatous disease: a review of 17 patients living in a region endemic for tuberculosis[END_REF][START_REF] Van Den Berg | Chronic granulomatous disease: the European experience[END_REF] (Supplementary information). All these observations are thus consistent with the Q231P and T178P CYBB mutations being responsible for XR-MSMD-2 in these two kindreds.

Normal NADPH oxidase activity in circulating phagocytes

CYBB encodes the β chain of flavocytochrome-b 558 , also known as gp91 phox or NOX2, an essential element of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex (PHOX) in phagocytes, such as granulocytes, monocytes, and macrophages. It is also expressed, but to a lesser extent, in other cells, such as dendritic cells and B lymphocytes. In all phagocytes of patients with CGD or "variant CGD", the production of reactive oxygen species (ROS) is inadequate. To solve the paradox of CYBB mutations in two kindreds with MSMD but apparently not CGD or "variant CGD" (Supplementary information), we therefore investigated in depth the respiratory burst in our patients bearing the Q231P or production was in the normal range, even at early time points, and proportional to the number of PMNs tested, even in the presence of catalase (Fig. 2a and Supplementary Fig. 2a and2b).

PMNs from the six patients tested also released H 2 O 2 normally in response to PMA [START_REF] Mohanty | A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative[END_REF] (Fig. 2b and Supplementary Fig. 2c). Following PMA treatment O 2 --dependent cytochrome-c reduction in monocytes from the six patients tested was normal (Fig. 2a and Supplementary Fig. 2d and2e). Similar amounts of H 2 O 2 were released after PMA activation of the monocytes from the five patients tested and healthy controls, contrasting with the defect seen in monocytes from CGD patients (Fig. 2b and Supplementary Fig. 2f). PMNs and monocytes from four heterozygous female subjects responded like control cells (Supplementary Fig. 2f). In addition, functional respiratory burst activity was assessed by flow cytometry, using dihydrorhodamine 123 (DHR) to measure intracellular levels of H 2 O 2 production. PMNs and monocytes from P4 (kindred A) and P5 (kindred B) stimulated with PMA were normal (Fig. 2c and Supplementary Fig. 2g).

Moreover, PMNs from P4 and P5 responded normally to milder activation involving priming with low concentrations of TNF, IL-1β or cytochalasin b, followed by fMLF stimulation (Fig. 2d and Supplementary Fig. 2h). Thus, blood PMNs and monocytes from the six patients bearing the Q231P or T178P CYBB allele tested showed a normal respiratory burst in peripheral blood PMNs and monocytes, as assessed by diverse assays of O 2 -production and H 2 O 2 release. Finally, we searched for subtle functional defects that might have been missed by the previous experiments, by evaluating the in vitro killing of Staphylococcus aureus by granulocytes from one patient from each kindred. Their granulocytes killed S. aureus normally, unlike granulocytes from patients with CGD, in which S. aureus is the leading pathogen (Fig. 2e). These findings confirm previous investigations of these patients' respiratory burst [START_REF] Bustamante | A novel X-linked recessive form of Mendelian susceptibility to mycobaterial disease[END_REF] and are consistent with the absence of clinical features typically associated with CGD and "variant CGD", including staphylococcal disease, even in affected adults of advanced age from kindreds A and B (Fig. 1a) [START_REF] Bustamante | A novel X-linked recessive form of Mendelian susceptibility to mycobaterial disease[END_REF] .

Impaired NADPH oxidase activity in macrophages

In turn, these findings raised the question of whether the two CYBB alleles were pathogenic at all in the two kindreds with MSMD. We thus investigated the cellular basis of mycobacterial disease in these patients by assessing the oxidative function of their monocytederived macrophages (MDMs) [START_REF] Nakagawara | Hydrogen peroxide metabolism in human monocytes during differentiation in vitro[END_REF][START_REF] Martinez | Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression[END_REF] . Indeed, tissue phagocytes, macrophages in particular, are the natural hosts of mycobacteria in the course of infection and disease. After 14 to 15 days of culture in the presence of M-CSF to generate MDMs, H 2 O 2 was clearly detectable when control MDMs were cultured for 16 to 18 hours with live BCG or PPD (purified protein derivative from M. tuberculosis) before PMA stimulation (Supplementary Fig. 3a). In the same conditions, macrophages from CGD patients, P1, P2, P3, and P4 (Q231P, kindred A), released no detectable H 2 O 2 . In similar conditions, but using interferon-γ (IFN-γ) rather than BCG or PPD, Q231P macrophages also did not respond normally to the PMA trigger (Supplementary Fig. 3a). M-CSF-differentiated macrophages from P5 (T178P, kindred B) displayed a milder phenotype, as they released low, or in some conditions normal amounts of H 2 O 2 (Supplementary Fig. 3a). We also studied formazan granule deposition due to the reduction of NBT, as a sensitive indicator of O 2 -production in individual macrophages derived from M-CSF-cultured monocytes. Only a very small fraction of BCG-or PPD-activated and PMA-triggered macrophages (less than 5%) from the four patients bearing the Q231P allele reduced NBT (Supplementary Fig. 3b). A larger fraction of macrophages from P5 bearing the T178P allele reduced NBT, but were strongly positive in less than 50%. As tissue macrophages from the patients were not available, we next tested MDMs derived in vitro in two conditions thought to reflect in vivo differentiation. We cultured MDMs with M-CSF alone for 7 days, and added LPS plus IFN-γ or IL-4. After 14 to 15 days of culture, MDMs (in any culture condition) from both P4 (kindred A) and P5 (kindred B)

were unable to release detectable H 2 O 2 , in contrast to control cells (Fig. 3, and Supplementary Fig. 3c). Thus, unlike blood granulocytes and monocytes, MDMs bearing the Q231P or the T178P CYBB allele, in multiple conditions of in vitro differentiation and conditions of stimulation, showed impairment of the respiratory burst, which was almost abolished for Q231P and severely impaired for T178P. We could not test the respiratory burst of the patients' macrophages in vivo or ex vivo. We characterized the patients' macrophage defect further, by assessing the growth of BCG within MDMs derived in vitro from two patients (P4 and P6) and ten healthy controls. As assessed by counting CFU, the patients' MDMs controlled BCG significantly less well than control MDMs on day 14 (p=0.06), and this difference was even more marked on day 21 (p=0.003) (Supplementary information and Supplementary Fig. 4). No such difference was observed in the presence of exogenous IFN-γ. These data provide a plausible mechanism for the co-segregation of the patients' CYBB mutation, their macrophage respiratory burst defect and their mycobacterial disease. As mycobacteria reside within macrophages in vivo, our in vitro experiments showing that MDMs in patients from each of the two kindreds display impairment of both the respiratory burst and of the control of BCG growth provide a plausible cellular basis for the occurrence of mycobacterial diseases in patients with XR-MSMD-2.

Impaired NADPH oxidase activity in EBV-B cells

CYBB is expressed in some B cells. Although irrelevant to the pathogenesis of XR-MSMD-2, as B cell-deficient patients are not prone to mycobacterial diseases [START_REF] Reichenbach | Mycobacterial diseases in primary immunodeficiencies[END_REF][START_REF] Conley | Primary B cell immunodeficiencies: comparisons and contrasts[END_REF] , we made use of this property to try to characterize the effects of the mutant CYBB alleles in Epstein Barr Virus (EBV)-transformed B-cell lines (EBV-B cells) [START_REF] Condino-Neto | NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system[END_REF] . Upon activation with PMA, EBV-B cells from 22 unrelated healthy individuals and nine male members of kindred A not carrying the Q231P CYBB mutation were capable of producing O 2 -(Fig. 4a and Supplementary Fig. 5). However, EBV-B cells from kindreds A and B (P1 to P7), like cells from XR-CGD patients, produced no detectable O 2 (Fig. 4a and Supplementary Fig. 5). We then measured the release of H 2 O 2 by EBV-B cells from P1 to P7 upon stimulation with various concentrations of PMA for various periods of time. None of the EBV-B cells from the seven patients released any detectable H 2 O 2 , like XR-CGD patients (Fig. 4b). Only 1 to 3% of the cells from the patients (P1 to P7) reduced NBT to formazan upon activation with PMA (Fig. 4c). EBV-B cells, MDMs therefore shared a similar cellular phenotype. We transiently transfected EBV-B cells from a healthy control, a patient with XR-CGD, P4 (kindred A), or P5 (kindred B) with plasmid-encoded wild-type or mutant CYBB, to determine whether the Q231P and T178P alleles were functionally hypomorphic. We then assessed the functional reconstitution of the respiratory burst in assays of NBT reduction. Forty-eight hours after transfection with wild-type CYBB, 10 to 13% of the cells from a patient with XR-CGD, P4, or P5 reduced NBT, but neither mock transfection nor transfection with either mutant CYBB had this effect (data not shown). We also established stable transfectants by means of a retroviral vector, and quantified the respiratory burst by assessing H 2 O 2 release. Stable transfection with WT CYBB allele complemented the defect in Q231P, T178P EBV-B cells indicating the restoration of a functional respiratory burst in Q231P, T178P

and XR-CGD EBV-B cells transduced with wild-type CYBB (Fig. 4d); however, no complementation of the cellular phenotype (no detectable respiratory burst) was observed after transduction of XR-CGD cells transduced with the Q231P or T178P allele (Fig. 4d). Thus, EBV-B cells from these patients had an impaired respiratory burst because they carried the Q231P or T178P CYBB allele. A lack of respiratory burst in peripheral B cells, by inference from EBV-B cells, would not account for MSMD [START_REF] Reichenbach | Mycobacterial diseases in primary immunodeficiencies[END_REF][START_REF] Conley | Primary B cell immunodeficiencies: comparisons and contrasts[END_REF] , but we demonstrate a causal relationship between two mutants hemizygous for Q231P or T178P CYBB mutations and impairment of the respiratory burst likely also applies to the patients' MDMs (M-CSF + IL4) (e.g. Fig. 3), which could account for the clinical phenotype of MSMD. In contrast, these CYBB alleles did not impair the respiratory burst of PMNs or monocytes (Fig. 2 and Supplementary Fig. 2), explaining the lack of the CGD or "variant CGD" phenotype in the patients.

Impaired gp91 phox expression in macrophages

The observed impairment of the functional respiratory burst in the patients' EBV-B cells and MDMs but not in PMNs or monocytes was investigated. The amount of CYBB mRNA was determined by real-time RT-PCR in four cell types from the five patients from two kindreds tested and was found to be similar to that in 5 healthy controls (Supplementary Fig. 6a and6b).

We next investigated the cell-specific impact of the germline Q231P and T178P CYBB mutations by assessing gp91 phox expression in the corresponding cells by immunoblotting with three specific antibodies (recognizing different epitopes on the gp91 phox protein) and increasing amounts of proteins. PMNs produced the mutant protein Q231P gp91 phox in diminished but detectable amounts (Fig. 5a, Supplementary Fig. 6c). Experiments with the antibody 54.1 suggested that PMNs produced almost normal amounts of T178P gp91 phox , whereas experiments with the other two antibodies suggested that this protein was produced in smaller amounts, perhaps reflecting the nature of the epitopes recognized (Fig. 5a and Supplementary Fig. 6c). Mutant Q231P protein levels were similarly low in monocytes, in which mutant T178P protein levels were also reduced but less affected (Fig. 5a,5b). Interestingly, the gp91 phox protein expression defect for the Q231P and T178P mutant gp91 phox was much more pronounced in MDMs (Fig. 5b), correlating with the greater defect in the respiratory burst in this cell type (Fig. 3 and Supplementary Fig. 3). However, a species migrating at 65 kDa was detected in the Q231P and T178P macrophage samples (Fig. 5b). This size corresponds to the high mannose gp65 precursor of gp91 phox , which may be more stable in macrophages compared to monocytes or macrophages, as has previously been reported for EBV-B cells 20,21 . This species is normally only transiently present in the endoplasmic reticulum prior to heme incorporation and heterodimer formation with p22 phox , which is followed by additional carbohydrate processing of the CYBB subunit in the Golgi to the mature gp91 phox of ≈ 91 kDa and ultimate targeting of flavocytochrome-b 558 to postbiosynthetic membrane compartments [START_REF] Yu | Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum[END_REF][START_REF] Deleo | Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly[END_REF][START_REF] Yu | Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits[END_REF][START_REF] Nakamura | Monoclonal antibody 7D5 raised to cytochrome b558 of human neutrophils: immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease, and their carrier mothers[END_REF][START_REF] Biberstine-Kinkade | Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox)[END_REF] . The gp65 species was also detected in an XR-CGD patient (Fig. 5b) with an L365P mutation in the flavin binding domain, but lacks gp91 phox expression and neutrophil oxidase activity [START_REF] Heyworth | Hematologically important mutations: X-linked chronic granulomatous disease (second update)[END_REF] . The presence of the gp65 intermediate suggests that these alleles are associated with impaired formation of the flavocytochrome b heterodimer, which is required for maturation of gp65 to gp91 phox . Finally, we did not have access to fresh tissue macrophages, but assessed gp91 phox production in the macrophages present in lymph node biopsy samples from two patients by immunohistochemistry (Supplementary Fig. 6d and6e). Impaired gp91 phox production was seen in macrophages from P4 (Q231P), as in lymph nodes from CGD patients, but not in healthy controls. Residual gp91 phox production was detected in tissue PMNs from P4. As expected, gp91 phox was detected in both PMNs and macrophages from P5 (T178P).

Altogether, these data indicate that the patients' defect of gp91 phox expression is more pronounced in macrophages compared to neutrophils and monocytes, and overall more severe for the Q231P CYBB allele.

Impaired gp91 phox expression in EBV-B cells

We also tested EBV-B cells from a healthy control, XR-CGD (in this case, carrying a complete deletion of the CYBB allele), and the Q231P and T178P patients, and the same cells transduced by wild-type or one of the mutant CYBB alleles (Fig. 5c). Clearly, the two mutant CYBB alleles are poorly expressed in EBV-B cells, in which the 91 kDa form of gp91 phox was barely detectable in cells derived from patients or following transduction of XR-CGD cells with retroviral vectors for expression of the mutant CYBB alleles. Again, a 65 kDa species corresponding to the high mannose precursor of the mature 91 kDa form of gp91 phox was detected for each of the Q231P and T178P mutant alleles, in contrast to EBV-B cells from an XR-CGD patient with a large deletion encompassing the CYBB gene (Fig. 5c). We also investigated the expression of the other components of the NADPH oxidase complex. Levels of p22 phox (which is encoded by CYBA and is normally bound to gp91 phox ) were reduced in PMNs and EBV-B cells, particularly in cells with the Q231P allele, paralleling gp91 phox expression (Supplementary Fig. 6f). Although expression of p22 phox in EBV-B cells is less affected by low or absent gp91 phox28 , p22 phox levels were still modestly reduced in EBV-B cells from P4 and P5 (Supplementary Fig. 6g) (providing further, indirect evidence for impaired gp91 phox expression in the patients' EBV-B cells). The p47 phox and p67 phox subunits were normally expressed in patients' PMNs and EBV-B cells (Supplementary Fig. 6f and6g). Reproducible differences in gp91 phox protein abundance were observed between cell types in six patients carrying either Q231P or T178P, with less gp91 phox in MDMs and EBV-B cells than in the patients' monocytes and PMNs, or as compared to in phagocytes and EBV-B cells from five controls. The Q231P CYBB mutation decreases the expression of the gp91 phox protein in vivo, ex vivo, and in vitro, in granulocytes, monocytes, macrophages and EBV-B cells, with steady-state levels of this protein being determined in part by the cell type, with a more pronounced impact in macrophages and EBV-B cells. The 178P gp91 phox protein is expressed at higher levels but follows an identical pattern according to the cell type. These expression data are consistent with the normal functional respiratory burst in the patients' PMNs and monocytes, contrasting with the functional defect in oxidant production by EBV-B cells and MDMs.

Impaired expression of flavocytochrome-b 558

We then analyzed the cell surface expression of the flavocytochrome-b 558 complex (gp91 phox and p22 phox ) on the plasma membrane by flow cytometry, using the monoclonal antibody 7D5, which recognizes residues 160 IKNP 163 and 226 RIVRG 230 on gp91 phox in the presence of p22 phox [START_REF] Yamauchi | Location of the epitope for 7D5, a monoclonal antibody raised against human flavocytochrome b558, to the extracellular peptide portion of primate gp91phox[END_REF][START_REF] Burritt | Phage display epitope mapping of human neutrophil flavocytochrome b558. Identification of two juxtaposed extracellular domains[END_REF] . Cell surface flavocytochrome-b 558 was clearly detectable in PMNs and monocytes from the two patients tested here, from two different kindreds, although at reduced levels for the Q231P allele (Fig. 5d and Supplementary Fig. 6h). By contrast, flavocytochrome-b 558 was barely detectable on EBV-B cells from seven patients tested among both kindreds (Fig. 5e).

Identical results were obtained by spectrophotometric measurements of reduced minus oxidized difference absorption spectra for PMNs and EBV-B cells (Supplementary Fig. 6i). Adherent MDMs were not suitable for these two assays and we did not have access to fresh tissue macrophages. Analysis of the cell surface expression of flavocytochrome-b 558 in PMNs, monocytes and EBV-B cells from the heterozygous female subjects revealed the presence of two discrete cell populations -those expressing and those not expressing flavocytochrome-b 558correlating with both the functional respiratory burst and the inactivated X chromosome (Supplementary Fig. 7a and 7b, and Supplementary information). Similar findings were obtained in NBT assays on MDMs from heterozygous female subjects (Supplementary Fig. 7c).

Thus, the Q231P and T178P mutations impair NADPH oxidase gp91 phox expression to a greater extent in MDMs and EBV-B cells than in PMNs and monocytes, revealing a profound cell type-specific effect on the flavocytochrome-b 558 complex, in terms of both surface and total expression. The relatively greater impact of the mutant alleles in MDMs and EBV-B cells may reflect cell-type-related differences in production of flavocytochrome-b 558 , which is higher in PMNs and monocytes compared to MDMs and EBV-B cells [START_REF] Cassatella | Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components[END_REF][START_REF] Condino-Neto | NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system[END_REF] . The much smaller amounts of mutant gp91 phox protein produced in EBV-B cells and in MDMs, and possibly in tissue macrophages, severely limit the capacity for the assembly of a functional NADPH oxidase complex, whereas the amounts produced in PMNs and monocytes, although smaller than normal, particularly for the Q213P allele, are sufficient to support assembly of adequate amounts of the functional enzyme complex to produce oxidants at normal levels (Fig. 2) [START_REF] Ezekowitz | Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon gamma[END_REF][START_REF] Ding | High-level reconstitution of respiratory burst activity in a human X-linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox[END_REF][START_REF] Ezekowitz | Restoration of phagocyte function by interferon-gamma in Xlinked chronic granulomatous disease occurs at the level of a progenitor cell[END_REF][START_REF] Zhen | Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox[END_REF] . Taken together, cell-type differences in the level of production of gp91 phox and the assembly of the flavocytochrome-b 558 heterodimer collectively account for the selective impairment of the respiratory burst in some cell types in the patients carrying the Q231P or T178P allele.

The mutant CYBB alleles are hypomorphic in CHO cells

We additionally characterized the two CYBB mutant alleles Q231P and T178P in a gp91 phox -deficient Chinese Hamster Ovary (CHO) epithelial cell line, in which gp91 phox can be expressed at higher levels upon transfection, allowing a fine study of its biochemical processing and association with p22 [START_REF] Yu | Biosynthesis of flavocytochrome b558 . gp91(phox) is synthesized as a 65-kDa precursor (p65) in the endoplasmic reticulum[END_REF][START_REF] Deleo | Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly[END_REF][START_REF] Yu | Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits[END_REF][START_REF] Casbon | Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes[END_REF][START_REF] Zhu | Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity[END_REF][START_REF] Biberstine-Kinkade | Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function[END_REF] . In CHO and other heterologous cells, as well as in EBV-B cells 20,21,28 , unassembled subunits are more stable than in granulocytes where these are rapidly degraded by the cytosolic proteasome [START_REF] Deleo | Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly[END_REF] . The detection of the CYBB-encoded gp65 intermediate in macrophages and EBV-B cells from each kindred (Fig. 5b,5c) suggested an underlying impairment in maturation to gp91 phox that occurs after formation of the flavocytochrome b heterodimer. In CHO cells transduced with retroviral vectors for expression of either the wildtype, Q231P or T178P CYBB alleles, similar amounts of the gp65 form were detected (Fig. 6a and 6b). The co-expression of wild-type p22 phox with wild-type CYBB increased cellular levels of mature 91 kDa gp91 phox (Fig. 6a and6b). With co-expression of p22 phox , mature T178P-gp91 phox was produced in almost normal amounts in the cell and on its surface, whereas Q231P-gp91 phox was produced in much smaller quantities; for both mutant alleles, substantial gp65 precursor was still present (Fig. 6a and6b). Thus, these findings suggest that there are no intrinsic problems in stability of the gp65 precursors encoded by two CYBB missense alleles but, rather, provide further evidence that the products of these alleles have reduced heterodimer formation with p22 phox and subsequent maturation to the mature gp91 phox , with the Q231P allele being more severely hypomorphic than the T178P allele, similar to the findings in patients' myeloid cells.

The mutant CYBB alleles are hypomorphic in PLB-985 cells

We then used an XR-CGD gp91 phox -deficient myelomonocytic PLB-985 cell line [START_REF] Zhen | Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox[END_REF] , which we also transduced with retroviral vectors for constitutive expression of wild-type-, Q231P-and T178P-gp91 phox . In the undifferentiated myelomonocytic cells, gp91 phox was detected in cells transduced with wild-type CYBB, whereas only small amounts of gp91 phox were detected following transduction with either of the mutant CYBB alleles, although the gp65 precursor was present (Fig. 6c). The mature T178P-gp91 phox was again produced in larger amounts than Q231P-gp91 phox . When the cell line was induced to differentiate into granulocytes, the two mutant alleles remained hypomorphic, with reduced levels of gp91 phox (Fig. 6c). As for fresh granulocytes from the patients, substantial respiratory burst activity relative to the amount of mature gp91 phox was supported by the mutant alleles in the granulocyte-differentiated cells (not shown). By contrast, gp91 phox was not expressed by the non transduced XR-CGD PLB-985 cell line, in which no respiratory burst was detectable. We also examined the levels of endogenous and of retrovirusexpressed gp91 phox following differentiation into monocyte-like or macrophage-like cells with either vitamin D [START_REF] Perkins | 1,25-Dihydroxyvitamin D3 induces monocytic differentiation of the PLB-985 leukemic line and promotes c-fgr mRNA expression[END_REF] or a combination of vitamin D and PMA [START_REF] Jitkaew | Induction of caspase-and reactive oxygen species-independent phosphatidylserine externalization in primary human neutrophils: role in macrophage recognition and engulfment[END_REF] , respectively. In wild-type PLB-985 cells, endogenous gp91 phox expression increased with either of these regimens (Fig. 6d). The level of wild-type gp91 phox expressed using the constitutively active retroviral vector was similar in undifferentiated cells and following either type of differentiation (Fig. 6d). In contrast, retrovirusmediated expression of gp91 phox harboring either the T178P or Q231P mutation was lower than that of wild-type gp91 phox , and decreased even further following macrophage differentiation (Fig. 6d). These experiments demonstrate that the Q231P and T178P alleles are hypomorphic in the myelomonocytic PLB-985 cell line. Although expression of the gp65 precursor of gp91 phox was relatively preserved with either of these two mutations, there was reduced, but not abolished expression, of gp91 phox itself, consistent with impaired flavocytochrome-b 558 formation, particularly following macrophage differentiation.

Discussion

We report here the first germline mutations in CYBB conferring the phenotype of XR-MSMD but not XR-CGD or "variant XR-CGD". Six otherwise healthy and maternally-related male adults with BCG-disease and another with bona fide tuberculosis had XR-MSMD-2, due to inheritance of the Q231P or T178P CYBB allele. The two missense mutations are intrinsically and severely hypomorphic in gp91 phox -deficient cells as diverse as EBV-B cells, CHO cells, and PLB-985 cells. CYBB is a novel Mendelian mycobacterial susceptibility gene, the seventh MSMD-and second XR-MSMD-causing gene identified, and the fifth tuberculosis-predisposing gene (after IFNGR1, IL12B, IL12RB1, and NEMO) [START_REF] Alcais | Tuberculosis in children and adults: two distinct genetic diseases[END_REF][START_REF]Inborn errors of IL-12/23-and IFN-gamma-mediated immunity: molecular, cellular, and clinical features[END_REF] . It is the first shown to affect the respiratory burst. The physiological links between CYBB and the formerly identified MSMD-causing and tuberculosispredisposing genes involved in the IL-12-IFN-γ circuit remain to be unraveled. The cellular phenotype of this defect is uniform and different from that of CGD and "variant CGD": the respiratory burst in MDMs or EBV-B cells from the seven patients tested was severely impaired, whereas their PMNs and monocytes showed no such dysfunction. The CYBB Q231P and T178P alleles are therefore severely hypomorphic in a cell-specific manner, providing an example of a human genetic illness resembling the phenotype of 'conditional knockout' mice, with the nuance that gene ablation in one of the two affected lineages also depends on cell differentiation (monocyte to macrophage). CYBB is a human gene for which alleles null in all cells (in particular all phagocytes, resulting in CGD) and now in selected cell types (in particular in macrophages, resulting in MSMD) have been identified and shown to produce different phenotypes. The mechanism underlying the B cell-and macrophage-specific effects of these germline mutations, which result in impaired but not absent flavocytochrome b 558 assembly, is related to cell-specific differences in gp91 phox expression, in the threshold of gp91 phox expression required for flavocytochrome-b 558 complex assembly, and in the threshold of assembled b 558 complex required for NADPH oxidase activity. The clear-cut dichotomy between cells with an entirely normal (PMNs, monocytes) respiratory burst and those with a severely impaired (MDMs, EBV-B cells) respiratory burst was remarkable. Further studies are required to test the impact of these two mutations on the respiratory burst of dendritic cells [START_REF] Savina | NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells[END_REF] .

Our study suggests that the respiratory burst in human tissue macrophages is critical for immunity against tuberculous mycobacteria. It is already clear from human patients with CGD [START_REF] Bustamante | BCG-osis and tuberculosis in a child with chronic granulomatous disease[END_REF] that the respiratory burst plays an important role in immunity to BCG and M. tuberculosis.

Intriguingly, these patients are not prone to infections with environmental, non-tuberculous mycobacteria, even species more virulent than BCG in other patients with MSMD, implying that the respiratory burst is redundant for the macrophage destruction of most non-tuberculous mycobacteria. The growth of BCG was enhanced in the patients' MDMs in vitro, further suggesting that there is a specific requirement for NADPH oxidase assembly to control tuberculous mycobacteria in human macrophages in vivo. Other pathways possibly disrupted by the impairment of the respiratory burst in macrophages, such as IL-12 production or antigen presentation, may also contribute to the pathogenesis of mycobacterial diseases. The role of the respiratory burst in anti-mycobacterial immunity in vivo and in vitro has not been firmly established in the mouse model, in which nitric oxide plays a much more important role [START_REF] Adams | Comparison of the roles of reactive oxygen and nitrogen intermediates in the host response to Mycobacterium tuberculosis using transgenic mice[END_REF][START_REF] Segal | The p47(phox-/-) mouse model of chronic granulomatous disease has normal granuloma formation and cytokine responses to Mycobacterium avium and Schistosoma mansoni eggs[END_REF][START_REF] Nathan | Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens[END_REF][START_REF] Macmicking | Nitric oxide and macrophage function[END_REF] .

Further genetic approaches [START_REF] Casanova | Primary immunodeficiencies: a field in its infancy[END_REF][START_REF] Alcais | Human genetics of infectious diseases: between proof of principle and paradigm[END_REF] may reveal the means by which the human respiratory burst controls tuberculous mycobacteria, and the role played by nitric oxide production against tuberculous or non tuberculous mycobacteria [START_REF] Macmicking | Nitric oxide and macrophage function[END_REF] . Conversely, our seven adult patients with MSMD (including a patient with tuberculosis) are clinically healthy and had no history of other granulomatous or infectious diseases. This strongly suggests that both the granulomatous disease, and the bacterial and fungal infections affecting patients with CGD or "variant CGD" reflect additional dysfunctions of granulocytes and/or monocytes, rather than macrophage defects alone [START_REF] Gordon | Monocyte and macrophage heterogeneity[END_REF][START_REF] Segal | How neutrophils kill microbes[END_REF] .

Methods

Patients

In kindred A, four male subjects presented a selective predisposition to mycobacterial disease [START_REF] Bustamante | A novel X-linked recessive form of Mendelian susceptibility to mycobaterial disease[END_REF] . Briefly, patient 1 (P1) suffered from bona fide tuberculosis and three other patients (P2, P3 and P4) developed infection by Mycobacterium bovis BCG vaccine. They suffered from no other severe infections. These four patients remain otherwise healthy. A maternal aunt (H1)

suffered from salpingitis and pulmonary tuberculosis.

All the members of kindred B are French and live in France. Three patients (P5, P6, and P7) suffered from BCG infection (see Supplementary information). No other unusual infections were reported in any of these three patients. Informed consent was obtained from all family members and our study was approved by the Necker and Rockefeller IRBs.

DNA techniques, PCR and sequencing

Genomic DNA was extracted with phenol/chloroform. The sequences of the primers used for PCR amplification of CYBB exons and cDNA are available upon request.

Preparation of PMNs, monocytes and MDMs

PMNs and monocytes were prepared from heparin-treated blood. Peripheral blood was subjected to dextran sedimentation and the buffy coat was centrifuged through Ficoll-Hypaque.

The remaining red blood cells were removed by hypotonic shock.

Monocytes were isolated by positive immunomagnetic depletion (CD14 MicroBeads, Miltenyi Biotec). MDMs were obtained from purified monocytes cultured in the presence of M-CSF (50 ng/ml; R&D Systems). We plated 2 x 10 4 monocytes or MDMs per well in complete RPMI 1640 medium (RPMI medium supplemented with 10% heat-inactivated pooled FBS (GIBCO BRL)) in 96-well plates (Nunc) and incubated them at 37°C in an atmosphere containing 5% CO 2 . One type of MDMs differentiated with M-CSF were obtained by adding, on day 7, Salmonella minesotta LPS (Sigma-Aldrich, 1 µg/ml) + IFN-γ (10 5 IU/ml, Imukin, Boehringer Ingelheim). The incubation was continued until day 16 to 17. Finally, other MDMs differentiated with M-CSF were obtained by adding IL-4 (50 ng/ml; R&D Systems) on day 7 and incubating until day 16 to 17 [START_REF] Martinez | Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression[END_REF] .

Superoxide assay

PMNs and monocytes were stimulated with 40 ng/ml of 4-beta-phorbol 12-beta-myristate 13-alpha-acetate (PMA, Sigma Aldrich) in the presence or absence of 140 IU/ml catalase (Sigma Aldrich). EBV-B cells were stimulated for 2 hours at 37°C. Superoxide release was assessed by the SOD-inhibitable cytochrome-c reduction assay, as previously reported [START_REF] Condino-Neto | NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system[END_REF] .

Hydrogen peroxide assay

Cells were stimulated with PMA at 37°C. EBV-B cells and measured hydrogen peroxide according to the kit manufacturer's (Amplex Red reagent: 10-acetyl-3,7-dihydroxyphenoxazine;

Molecular Probes, Invitrogen). The hydrogen peroxide released was quantified with Victor TM X4 (Perkin Elmer). MDMs were cultured for 16 to 18 hours with live BCG (Mycobacterium bovis BCG, Pasteur substrain at a multiplicity of infection of 10:1), PPD (1 mg/ml, Tuberculin PPD Batch RT49, Statens Serum Institute, Denmark) or IFN-γ (10 5 IU/ml; Imukin, Boehringer Ingelheim), then washed in KRPS and activated by incubation for 30 min with PMA (400 ng/ml) or left unactivated.

DHR assay

Peripheral leukocytes were incubated with dihydrorhodamine 123 (DHR; Sigma Aldrich) at 37°C for 5 minutes in the presence of catalase (1,300 IU/ml; Sigma Aldrich), and after activation with PMA for 30 minutes. The leukocytes were first treated with TNF-α (20 ng/ml; R&D Systems), IL-1β (40 ng/ml; R&D Systems) or cytochalasin b (10 µg/ml; Sigma-Aldrich)

for 10 minutes at 37°C and were then stimulated with fMLF (80 µg/ml; Sigma Aldrich) for 20 minutes. 10,000 or 5,000 events were recorded by flow cytometry on a FACScan or FACS Canto II machine (Becton Dickinson).

NBT reduction

Coverslips (10 mm) were placed in 24-well plates (Nunc), and 2 x 10 5 MDMs per well were plated in complete RPMI 1640 medium and incubated at 37°C under an atmosphere containing 5% CO 2 for 16 to 17 days. After 18 hours of incubation with live BCG (10:1) or PPD

(1 mg/ml), the MDMs were or were not exposed to PMA (400 ng/ml), in the presence of NBT (200 µg/ml, Sigma Aldrich) in HBSS at 37°C for 1 hour, and were then counterstained by incubation with safranin (1%) for 10 minutes. The percentage of NBT-positive cells (blue formazan dye) was determined by light microscopy (QCapture), scoring 100 cells. EBV-B cells were activated by incubation with PMA for 2 hours at 37°C.

Immunoblotting

Cell lysates were analyzed by immunoblotting as described [START_REF] Zhu | Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity[END_REF] . The membrane was incubated overnight with antibodies against gp91 phox (BD Biosciences-clone 53,54.1 and 7A2 ).

For p22 phox (SC-20781), p47 phox (SC-14015), p67 phox (SC-7663), and GAPDH (SC-25778), we used polyclonal antibodies from Santa Cruz.

Flow cytometric detection of flavocytochrome-b 558

Cells were incubated with 7D5 antibody (MBL Co., Ltd., Japan) or isotypic control (mouse IgG1, BD PharMingen) at a concentration of 5 µg/ml, and at 4°C for 30 minutes. Cells were analyzed with a FACScan flow cytometer or FACS Canto II (Becton Dickinson). 10,000 or 5,000 cells from each sample were collected and analyzed with Cell Quest software (BD Pharmingen).

Microbicidal assay with Staphylococcus aureus

Granulocytes were diluted in RPM and Staphylococcus aureus (Calbiochem) in RPMI supplemented with 10 % pooled human serum. After preopsonisation of the bacteria, 2 x 10 6 granulocytes/ml were mixed with 2 x 10 7 /ml S. aureus CFU/ml, in a 14 ml round-bottomed polypropylene tube (Falcon). The tubes were incubated, at 37°C for 30 minutes at 100 x g. The cell suspensions were then centrifuged at 160 x g and 4°C for 10 minutes. The supernatant was removed and the cell pellets were resuspended in 1 ml of RPMI with 100 µg/ml of gentamicin and incubated at 37°C for 4 times at 100 x g. The samples were centrifuged at 160 x g at 4°C for 10 minutes. The granulocytes were lysed with 200 µl of 0.1% Triton. CFU bacteria was counted by plating serial dilutions after incubation.

Retroviral transduction of EBV-B, CHO, and PLB-985 cells

A human cDNAs encoding the gp91 phox WT, gp91 phox T178P and gp91 phox Q231P were cloned into pMSCVpuro (Clontech). Retroviral vectors were packaged using the Pantropic Retroviral Expression System (BD Clontech). EBV-B cells, CHO22 cells generated for stable expression of untagged p22 phox and promyelocytic PLB-985 harboring a disrupted CYBB (PLB X-CGD cells) were transduced with MSCVPuro-gp91 phox WT, MSCVPuro-gp91 phox Q231P or MSCVPuro-gp91 phox T178P retrovirus. CHO-K1 cell lines utilized in these studies were generated previously [START_REF] Casbon | Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes[END_REF][START_REF] Biberstine-Kinkade | Mutagenesis of p22(phox) histidine 94. A histidine in this position is not required for flavocytochrome b558 function[END_REF] . For neutrophil and monocyte-macrophage differentiations, PLB-985 or PLB XR-CGD cells were cultured as previously described [START_REF] Zhen | Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox[END_REF][START_REF] Price | Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system[END_REF] .

I

Figure 1 .

 1 Figure 1. Q231P and T178 CYBB mutations in an X-linked recessive form of MSMD-

Figure 2 .

 2 Figure 2. Evaluation of NADPH oxidase activity in PMNs and monocytes. Superoxide

Figure 3 .

 3 Figure 3. Evaluation of NADPH oxidase activity in MDMs. Release of hydrogen

Figure 4 .

 4 Figure 4. Evaluation of NADPH oxidase activity in EBV-B cells. a, Superoxide production was measured by cytochrome-c reduction. 10 6 EBV-B cells from healthy controls

Figure 5 .

 5 Figure 5. Expression of gp91 phox and flavocytochrome-b 558 in the patients' cells.

Figure 6 .

 6 Figure 6. Expression and function of mutant gp91 phox in cell lines. a, CHO and CHO22cells were transduced with pMSCVPuro-gp91 phox WT, pMSCVPuro-gp91 phox T178P and pMSCVPuro-gp91 phox Q231P retroviral particles, and then selected with puromycin. Cell lysates of the transduced cells, CHO, CHO22 and CHO phox cells were evaluated for gp91 phox (mAb 54.1), p22 phox (mAb NS5) to detect the gp91 phox maturation and p22 phox expression. An antibody against β-actin was used as a protein loading control. b, Cell surface gp91 phox detected with 7D5 antibody using flow cytometry in CHO22 and CHO22 expressing gp91 phox WT, gp91 phox Q231P and gp91 phox T178P. c, and d, PLB X-CGD cells were transduced with pMSCVPuro-gp91 phox WT, pMSCVPuro-gp91 phox T178P or pMSCVPuro-gp91 phox Q231P retroviral particles, and then

  Figure 1

Acknowledgments

We warmly thank family members for their kind willingness to participate in this study. We thank J. Curnutte for EBV-B cells from CGD controls, D. Roos for mAb449, F. Morel for mAb7A2 and M. Quinn for mAb54.1. We thank all members of the two branches of the laboratory of Human Genetics of Infectious Diseases for discussions and Tony Leclerc, Yoann Rose, Mbia Kezadi, and Natalie Stull for technical assistance. Jacinta Bustamante was supported

by INSERM, EU grant NEOTIM EEA05095KKA, and March of Dimes (RO5050KK). A. Condino-Neto was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). This work was supported by Fondation BNP-Paribas, Fondation Schlumberger, Institut Universitaire de France, the ANR, The Rockefeller University Center for Clinical and Translational Science grant number 5UL1RR024143-03, The Rockefeller University, EU grants HOMITB HEALTH-F3-2008-200732 and NEOTIM EEA05095KKA, NIH grants DK54369 (P.E.N.) and HL045635 (M.C.D.), and the Riley Children's Foundation (M.C.D.). Jean-Laurent Casanova was an International Scholar of the Howard Hughes Medical Institute (2005-2008).

Competing interests statement

The authors declare that they have no competing financial interests.


Mathieu Lafourcade

Thomas Larrieu

Susana Mato

Anais Duffaud

Marja Sepers

Isabelle Matias

Veronique De Smedt

Virginie Labrousse

Lionel Brétillon

Carlos Matute

Lionel Bretillon

Rafael Rodríguez-Puertas

Sophie Layé

Olivier J Manzoni

Nutritional Omega-3 deficiency abolishes endocannabinoid mediated neuronal functions

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

Human deficiency diseases are due to a lack of essential nutrients in the diet. In developed countries, inexpensive high-calorie high fat foods are also low in essential nutrients, and malnutrition often is a corollary of obesity. In the United States about 30 percent of all pregnancies are carried by obese women but the functional long-term consequences of maternal malnutrition on the brains and behavior of their progeny are mostly unknown.

Lipid molecules are the building blocks of the central nervous system (CNS). In contrast to other tissues, the CNS and retina are enriched in polyunsaturated fatty acids (PUFAs): arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3). These long chain PUFAs are indispensable to the normal development and function of the CNS [START_REF] Bourre | Dietary omega-3 Fatty acids and psychiatry: mood, behaviour, stress, depression, dementia and aging[END_REF] . Linoleic acid (18:2n-6, LA, the precursor of AA) and αlinolenic acid (18:3n-3, ALA, the precursor of DHA) are not synthesized de novo by mammals and a balanced diet containing appropriate amounts of these precursors is obligatory to maintain sufficient brain levels of long chain PUFAs [START_REF] Bourre | Dietary omega-3 Fatty acids and psychiatry: mood, behaviour, stress, depression, dementia and aging[END_REF][START_REF] Rapoport | Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver[END_REF] . Brain and retina DHA decreasee resulting from nutritional deficiency or aging are associated with reduced cognitive ability, increased emotional behavior and decreased vision [START_REF] Lauritzen | The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina[END_REF] .

Despite their high caloric contents, western diets are poor in essential nutrients and notorious for their low levels of n-3 and high levels of n-6 PUFAs [START_REF] Simopoulos | Evolutionary aspects of the dietary omega-6:omega-3 fatty acid ratio: medical implications[END_REF] . Fetuses and newborns exclusively rely on their mother's diet for their PUFAs intake. Indeed, major structural fatty acids such as DHA that cannot be efficiently metabolized by developing babies must be received from the mother through the placenta during pregnancy and through breast milk during nursing. Finally, the deleterious consequences of maternal malnutrition on the progeny are further aggravated by a lifetime of nutrient poor diet.

On the basis of the epidemiological and clinical data linking n-3 PUFAs deficiency and mood disorders [START_REF] Parker | Omega-3 fatty acids and mood disorders[END_REF] , we hypothesized that lifelong malnutrition may influence synaptic functions in brain areas controlling mood.

Here, we used a specific diet to mimic lifelong n-3/n-6 imbalance of essential PUFAs in mice and discovered that reducing n-3 levels dramatically diminished the synaptic and behavioral functions of the cannabinoid CB 1 R.

Results:

Impact of n-3 dietary deficiency on brain lipids:

To evaluate the consequences of maternal malnutrition on the brains of their offspring, mice C57BL6/J females were fed with a diet containing 6% fat in the form of African peanut oil rich in 18:2n-6 called "n-3 deficient diet" or an isocaloric diet composed of African peanut oil and rapeseed oil naturally rich in 18:3n-3 called "n-3 diet" throughout gestation and lactation [START_REF] Mingam | Uncoupling of interleukin-6 from its signalling pathway by dietary n-3-polyunsaturated fatty acid deprivation alters sickness behaviour in mice[END_REF] . After weaning, the offspring was submitted to the same diet throughout the rest of their life to model a lifetime of malnutrition.

We found that lifelong dietary imbalance in the ratio of n-3/n-6 PUFAs had a major impact on total brain PUFAs levels (Fig. 1a-b). The n-3 deficient diet did not change saturated or mono-unsaturated fatty acids levels (not shown) but it specifically decreased the brain levels of the major n-3 PUFAs such as DHA (22:6n-3), docosapentaenoic acid (22:5n-3, DPA) and eicosapentaenoic acid (20:5n-3, EPA), while at the same time it increased n-6 PUFAs such as docosapentanoic acid (22:5n-6, DPA; Fig. 1) [START_REF] Mingam | Uncoupling of interleukin-6 from its signalling pathway by dietary n-3-polyunsaturated fatty acid deprivation alters sickness behaviour in mice[END_REF] .

n-3 deficiency ablates endocannabinoid synaptic plasticity:

How could synaptic functions be perturbed by those changes in brain PUFA? Dietary PUFAs disproportion modifies the brain lipid biochemistry 1 , impacts on monoaminergic neurotransmitter, glutamate receptors and impairs rodents' behavior (reviewed by [START_REF] Bourre | Dietary omega-3 Fatty acids and psychiatry: mood, behaviour, stress, depression, dementia and aging[END_REF][START_REF] Chalon | Omega-3 fatty acids and monoamine neurotransmission[END_REF] ). Furthermore, convergent epidemiological and clinical studies have linked deficits in dietary n-3 PUFAs and mood disorders [START_REF] Parker | Omega-3 fatty acids and mood disorders[END_REF] .

The endocannabinoid (eCB) system is in a unique position to link food lipids, synaptic activity and behavior. The two principal eCBs, anandamide (AEA) and 2arachidonoylglycerol (2-AG), are signaling lipids produced from membrane longchain fatty acids in response to neuronal activity that bind a G-protein coupled receptor (GPCR) named CB 1 receptor (CB 1 R) [START_REF] Mackie | Signaling via CNS cannabinoid receptors[END_REF] . The eCB system is a major actor of synaptic plasticity [START_REF] Heifets | Endocannabinoid signaling and long-term synaptic plasticity[END_REF] and its deregulation has been postulated to contribute to the etiology of mood disorders [START_REF] Leweke | Cannabis and psychiatric disorders: it is not only addiction[END_REF][START_REF] Lutz | Endocannabinoid signals in the control of emotion[END_REF][START_REF] Hill | The therapeutic potential of the endocannabinoid system for the development of a novel class of antidepressants[END_REF] .

The prefrontal cortex (PFC) is normally implicated in executive tasks and reward and has recently been proposed to participate to emotional behavior and the pathophysiology of depression [START_REF] Krishnan | The molecular neurobiology of depression[END_REF][START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF] . Thus we focused our attention on the PFC in the present study.

After verifying that n-3 deficiency modified PUFAs levels in the PFC (Fig. 1c-d), we evaluated how dietary PUFAs influence eCB-mediated synaptic plasticity in our animal model. In PFC slices prepared from mice that had received a n-3 diet, tetanic stimulation induced a robust eCB-mediated long-term depression (eCB-LTD) of excitatory synapses onto layer V/VI pyramidal neurons within the prelimbic area (PrPFC) (Fig. 2a) [START_REF] Lafourcade | Molecular components and functions of the endocannabinoid system in mouse prefrontal cortex[END_REF] . This form of synaptic plasticity normally mediated by eCB was totally ablated in mice fed with the n-3 deficient diet (Fig. 2a,b).

The medial PrPFC sends major excitatory projections to the accumbens, an anatomical substrate for reward and motivation. Noteworthy, eCB-LTD was initially described in the accumbens [START_REF] Robbe | Endogenous cannabinoids mediate long-term synaptic depression in the nucleus accumbens[END_REF] and synaptic dysfunctions in the accumbens may participate to the etiology of mood disorders [START_REF] Krishnan | The molecular neurobiology of depression[END_REF][START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF] .

As in the PrPFC, eCB-LTD was lacking in the accumbens of n-3 deficient mice (Fig. 2c), indicating that n-3 deficiency may abolishe eCB-mediated plasticity in different areas.

n-3 deficiency does not perturb other types of synaptic plasticity:

We next evaluated if the nutritional-induced impairment of eCB-LTD had generalized to other forms of plasticity at PrPFC synapses.

Input-output relationships were statistically indistinguishable in the two mice groups, ruling out the possibility that the incapacity to trigger eCB-LTD in n-3 deficient mice was caused by decreased excitability of glutamatergic PrPFC synapses (Supplementary Fig. 1). The ratio between AMPAR mediated and NMDAR mediated components of evoked excitatory currents is an indicator of previous longterm synaptic plasticity [START_REF] Kauer | LTP: AMPA receptors trading places[END_REF][START_REF] Hayton | Targetspecific encoding of response inhibition: increased contribution of AMPA to NMDA receptors at excitatory synapses in the prefrontal cortex[END_REF] . This index was indistinguishable between the two groups of mice, showing that the conventional long-term potentiation or LTD mechanisms have not been expressed in the PrPFC following n-3 deficiency (Fig. 3a).

To determine whether n-3 deficiency acted on the transduction pathways downstream of presynaptic CB 1 R, we took advantage of the existence of another presynaptic type of LTD triggered by mGluR2/3 receptors [START_REF] Robbe | Role of p/q-Ca2+ channels in metabotropic glutamate receptor 2/3-dependent presynaptic long-term depression at nucleus accumbens synapses[END_REF] and mediated by the same presynaptic transduction pathways [START_REF] Robbe | Role of p/q-Ca2+ channels in metabotropic glutamate receptor 2/3-dependent presynaptic long-term depression at nucleus accumbens synapses[END_REF][START_REF] Mato | Role of the cyclic-AMP/PKA cascade and of P/Q-type Ca++ channels in endocannabinoid-mediated long-term depression in the nucleus accumbens[END_REF] . The LTD induced by the mGluR 2/3 agonist LY379268 (100 nM) was normal in the n-3 deficient group (Fig. 3b), strongly suggesting that n-3 deficiency had no effect on the signaling cascades downstream of mGluR 2/3 or CB 1 R.

Taken together these data show that the deleterious effects of the unbalanced diet are not due to unspecific or generalized alteration of synaptic transmission and plasticity, but rather to the specific ablation of eCB-mediated synaptic plasticity.

n-3 deficiency desensitizes and uncouples CB 1 receptors:

A number of different mechanisms may explain the observed lack of eCB-plasticity in n-3 deficient mice. Altered PUFA environment may directly impairs CB 1 Rs functionality. Previous work from our laboratory has demonstrated the exquisite sensitivity of the eCB system to agonist-induced desensitization of CB 1 R in vivo and in vitro [START_REF] Mato | A single in-vivo exposure to delta 9THC blocks endocannabinoid-mediated synaptic plasticity[END_REF][START_REF] Mikasova | Altered surface trafficking of presynaptic cannabinoid type 1 receptor in and out synaptic terminals parallels receptor desensitization[END_REF] . To interrogate the effects of n-3 deficiency on CB 1 Rs presynaptic functions we built dose-response curves for the CB agonist CP55,940 in the two groups. In n-3 diet mice, the CB agonist inhibited synaptic transmission similarly to what we previously reported in mice fed with standard chow [START_REF] Lafourcade | Molecular components and functions of the endocannabinoid system in mouse prefrontal cortex[END_REF] . In striking contrast, there was a massive reduction in the maximum inhibitory effects of the CB agonist in the n-3 deficient group (Fig. 4a). We tested whether this impairment was associated to reduced coupling of CB 1 Rs to their effector G i/o proteins using [ 35 S]GTPγS autoradiography [START_REF] Mato | Presynaptic homeostatic plasticity rescues long-term depression after chronic Delta 9tetrahydrocannabinol exposure[END_REF] . Basal [ 35 S]GTPγS binding levels in PrPFC were similar between n-3 and n-3 deficient mice (data not shown). However, the ability of the cannabinoid agonist WIN55,212-2 (WIN) to stimulate [ 35 S]GTPγS binding was significantly reduced in PrPFC layers V-VI of n-3 deficient mice (Fig. 4b and Supplementary Fig. 2).

To determine if n-3 deficient diet impaired CB 1 R coupling in different areas, we analyzed WIN-induced stimulation of [ 35 S]GTPγS binding in the motor cortex within the same brain sections (Fig. 4b). We detected no difference in the ability of the CB agonist to stimulate [ 35 S]GTPγS binding in motor cortex between n-3 and n-3 deficient mice. Thus, brain region specific uncoupling of CB 1 R to G i/o proteins may underlie the impairment of CB 1 R function in PrPFC of n-3 deficient mice. Desensitization of CB 1 Rs at the G i/o protein level may ensue from receptor downregulation [START_REF] Martin | Signaling pathways involved in the development of cannabinoid tolerance[END_REF] Thus, the effect of n-3 deficient diet on CB 1 R expression in the PFC was analyzed by means of receptor autoradiography and western blot. The density of CB 1 Rs, measured by specific binding of [ 3 H]CP,55940 in the prelimbic and motor cortices, was similar between n-3 and n-3 deficient mice (Fig. 4c). Consistently, total protein levels of CB 1 R in PFC remained unaffected by the diet (0.090 ± 0.005, n=8 and 0.085±0.009, n=8 in n-3 and n=9 in n-3 deficient diet, respectively).

Collectively, our data strongly argue against the idea that reduced CB 1 R expression levels underlie diminished CB 1 R function in n-3 deficient mice (Fig. 4d).

Effects of n-3 deficiency on circulating and synaptic eCB levels:

Because membrane bound PUFAs are the precursors of eCB, dietary PUFAs could elevate circulating eCB levels that in turn would saturate and/or desensitize CB 1 R 25 .

However, the brain bulk levels of AEA and 2-AG were similar in both groups (Supplementary Fig. 3). The apparent discrepancy with previous data showing interactions between diets and circulating eCBs could be due to homeostatic adaptations restoring normal eCB during a lifetime of n-3 deficiency [START_REF] Berger | Anandamide and diet: inclusion of dietary arachidonate and docosahexaenoate leads to increased brain levels of the corresponding Nacylethanolamines in piglets[END_REF][START_REF] Matias | Effect of polyunsaturated fatty acids on endocannabinoid and N-acylethanolamine levels in mouse adipocytes[END_REF][START_REF] Watanabe | n-3 Polyunsaturated fatty acid (PUFA) deficiency elevates and n-3 PUFA enrichment reduces brain 2arachidonoylglycerol level in mice[END_REF] . An alternative explanation is that bulk measurements cannot accurately report the eCB concentration near synapses. To estimate the synaptic levels of eCB we compared the effects of the specific CB1R antagonist AM251 on baseline EPSCs in both groups. Bath application of the selective CB 1 R antagonist AM-251 (4 µM, 30 min) did not increase baseline synaptic transmission in the PrPFC of n-3 diet mice (99.31% of control, n=13). In contrast, the same treatment caused a small but significant increase in the n-3 deficient group (112.5%, n=11; unpaired t-test, p = 0.0358). Thus, as a whole, the data suggest that, in n-3 deficient mice, the inability to induce eCB plasticity results from the partial occupation of CB 1 R by enhanced eCB levels near synapses, combined to CB 1 R desensitization.

Behavioral correlates of diet-induced CB 1 R functional antagonism:

What are the behavioral correlates of the diet-induced CB 1 R functional antagonism and the associated ablation of eCB mediated synaptic plasticity? The most obvious denominator between n-3 PUFAs, CB 1 R and PrPFC/accumbens is their involvement in mood disorders and emotional behaviors [START_REF] Parker | Omega-3 fatty acids and mood disorders[END_REF][START_REF] Leweke | Cannabis and psychiatric disorders: it is not only addiction[END_REF][START_REF] Krishnan | The molecular neurobiology of depression[END_REF][START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF][START_REF] Vinod | Role of the endocannabinoid system in depression and suicide[END_REF] . Indeed, rats submitted to n-3 PUFA deficiency during 15 weeks from weaning 31 display higher depressive-like symptoms in the Porsolt forced swimming test (FST) [START_REF] Porsolt | Rodent models of depression: forced swimming and tail suspension behavioral despair tests in rats and mice[END_REF] We confirmed these data [START_REF] Porsolt | Rodent models of depression: forced swimming and tail suspension behavioral despair tests in rats and mice[END_REF] in n-3 deficient mice. There was an increase in the time spent immobile (Fig. 5a) and a reduction of the swimming time (Fig. 5b) in n-3 deficient diet mice compared to the n-3 diet group. We extended a previous report [START_REF] Demar | One generation of n-3 polyunsaturated fatty acid deprivation increases depression and aggression test scores in rats[END_REF] and showed that the tricyclic antidepressant imipramine reversed immobility in both groups (Fig. 5a,b).

Exploratory behaviors with emotional load (open-field and social exploration) are impaired in CB 1 R deleted mice [START_REF] Haller | Context-dependent effects of CB1 cannabinoid gene disruption on anxiety-like and social behaviour in mice[END_REF][START_REF] Uriguen | Impaired action of anxiolytic drugs in mice deficient in cannabinoid CB1 receptors[END_REF] . We found that n-3 deficiency decreases the number of social exploration (i.e. new congener investigation, 77.1 ± 1.3 n = 28 and 60.2 ± 1.9 n=16, in n-3 diet and n-3 deficient mice, respectively; p < 0.001) while increasing the number of litter scratching, an index of anxiety (16.9 ± 1.4 and 44.2 ± 3.7 in n-3 diet and n-3 deficient mice respectively; p < 0.001) with no significant effect on locomotion (2958.3 ± 61.9 cm n-3 diet and 2945.8 ± 73.0, n-3 deficient diet n=16).

Furthermore, in the open-field test, n-3 deficient mice spent significant less time in the center on the arena (11.0 ± 0.6 % of total time, n=26) than n-3 diet mice (8.0 ± 0.5 % of total time, n=16, p < 0.01). In addition, thigmotaxis, the tendency to remain close to the wall, a widely accepted index of anxiety [START_REF] Simon | Thigmotaxis as an index of anxiety in mice. Influence of dopaminergic transmissions[END_REF] , was significantly higher in n-3 deficient mice compared to n-3 mice (88.8 ± 0.6 % and 91.8 ± 0.5 % of total time, n-3 diet and n-3 deficient mice respectively; p < 0.01). Increased despair behavior was not linked to impaired total motor locomotion (total distances explored were 2814.1 ± 57.4 cm and 2757.5 ± 135.1 cm, in n-3 diet and n-3 deficient diet groups, respectively).

We then evaluated whether the behavioral effect of a CB agonist, was altered in n-3 deficient mice. We tested the effect of the administration of the cannabinoid agonist WIN (0.01-1 mg/kg) on emotional behavior, measured in the open-field test [START_REF] Bambico | Cannabinoids elicit antidepressant-like behavior and activate serotonergic neurons through the medial prefrontal cortex[END_REF] , immediately after the injection (Fig. 5c-d). In n-3 mice, WIN (0.1 mg/kg) reduced the time spent in the center of the arena and increased thigmotaxis. This anxiogenic effect of WIN (0.1 mg/kg) was ablated in n-3 deficient mice, corroborating our biochemical and synaptic data showing impaired CB 1 R functionality in n-3 deficient mice.

Discussion:

There is a considerable interest in understanding how food participates to health and disease. The obesity epidemic illustrates how food, in particular low-cost food with high sugar and fat contents and low essential nutrients levels, shapes our bodies and determines our general health status.

PUFA precursors of the n-3 or n-6 families are essential nutriments that cannot be generated de novo in mammals. They exist in plants as precursors 18:2n-6, LA and 18:3n-3, ALA and are metabolized by elongations and desaturations into AA, EPA and DHA in mammals [START_REF] Rapoport | Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver[END_REF] . The conversion of these precursors into long-chain PUFAs is mostly hepatic, although other organs such as the brain express the necessary enzymatic equipment [START_REF] Rapoport | Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver[END_REF] . Since the two series of PUFAs compete for their biosynthetic enzymes, and because they have distinct physiological properties, the dietary n-6/n-3 ratio is of fundamental importance.

In contrast to hunting and gathering food products rich in n-3 PUFAs that composed human diet in the past, modern diets are notoriously poor in these nutrients. Most notably, since the dawn of the industrial revolution, the ratio of n-6/n-3 PUFAs in the diet has steadily increased from 1 to almost 15 within industrialized countries, leading to a significant deficiency in n-3 PUFAs 4 . Modern western diets are characterized by their high saturated fat and sugar contents associated with low n-3 PUFAs levels. Such dietary customs critically contribute to the high prevalence of chronic diseases such as obesity and metabolic disorders in westernized countries.

Obesity is associated with psychological morbidity, including major depression [START_REF] Evans | Mood disorders in the medically ill: scientific review and recommendations[END_REF] , yet the underlying pathophysiological mechanisms remain poorly understood.

Here, we used a specific diet to mimic lifelong n-3/n-6 imbalance of essential PUFAs in mice. We discovered that reducing the n-3 levels dramatically reduces the function of the most abundant GPCR of the CNS, the cannabinoid CB 1 R receptor.

At the mechanistic level, our data revealed that CB 1 R functional antagonism [START_REF] Schlosburg | Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system[END_REF] results from its region-specific uncoupling from effector G i/o -proteins, and its partial occupation by enhanced eCB levels near synapses. This impairment has functional consequences and is reflected by behavioral modifications: the anxiogenic effect of a cannabinoid agonist, WIN, does not occur in n-3 deficient mice.

Hence, dietary n-3 deficiency modifies CB 1 R functions in a way reminiscent to that of classical agonist desensitization [START_REF] Mato | A single in-vivo exposure to delta 9THC blocks endocannabinoid-mediated synaptic plasticity[END_REF][START_REF] Mikasova | Altered surface trafficking of presynaptic cannabinoid type 1 receptor in and out synaptic terminals parallels receptor desensitization[END_REF][START_REF] Martin | Signaling pathways involved in the development of cannabinoid tolerance[END_REF] . Although we were not able to detect changes in circulating eCB levels in the brains of the adult mice used in the physiology and behavioral experiments, one cannot exclude the possibility that durable modifications in CB 1 R coupling were caused by transient but sizeable elevation of eCB levels at earlier stages. Previous reports of enhanced eCBs levels, in particular 2-AG (the natural full agonist of CB 1 R) in response to short-term diets, as well as on the ability of chronically elevated levels of this eCB to induce functional antagonism of brain CB 1 R via uncoupling from G i/o proteins [START_REF] Schlosburg | Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system[END_REF] , favor such a scenario [START_REF] Berger | Anandamide and diet: inclusion of dietary arachidonate and docosahexaenoate leads to increased brain levels of the corresponding Nacylethanolamines in piglets[END_REF][START_REF] Matias | Effect of polyunsaturated fatty acids on endocannabinoid and N-acylethanolamine levels in mouse adipocytes[END_REF][START_REF] Watanabe | n-3 Polyunsaturated fatty acid (PUFA) deficiency elevates and n-3 PUFA enrichment reduces brain 2arachidonoylglycerol level in mice[END_REF] .

Functionally, an important consequence of CB 1 R desensitization is to abolish eCBmediated synaptic plasticity in two brain regions implicated in emotional behavior and mood disorders, the PrPFC and the accumbens [START_REF] Krishnan | The molecular neurobiology of depression[END_REF][START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF] . Our data strongly suggest that the deleterious impact of n-3 deficiency on eCB plasticity does not generalize to other forms of synaptic plasticity, including mGluR2/3-LTD that shares the signaling pathway of CB 1 R-LTD. Furthermore, in contrast to what we observed following in vivo agonist-desensitization of CB 1 R, there is no homeostatic rescue of LTD by autocrine activation of presynaptic mGluR2/3 [START_REF] Samat | Rimonabant for the treatment of obesity[END_REF] .

Preclinical data point to an important role of CB 1 R and the eCB system in the control of mood and emotionality [START_REF] Leweke | Cannabis and psychiatric disorders: it is not only addiction[END_REF][START_REF] Lutz | Endocannabinoid signals in the control of emotion[END_REF][START_REF] Hill | The therapeutic potential of the endocannabinoid system for the development of a novel class of antidepressants[END_REF][START_REF] Vinod | Role of the endocannabinoid system in depression and suicide[END_REF] . In many aspects, the emotional disturbances observed in n-3 PUFAs deficient mice resemble the ones measured in CB 1 R null mice: increased thigmotaxis in the open-field test and lower social interactions were measured in CB 1 R null mice as compared to wild-type littermates [START_REF] Jacob | Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: role of glutamatergic transmission[END_REF] . In agreement, the floating time measured in the FST was higher in CB 1 R null mice [START_REF] Steiner | Impaired cannabinoid receptor type 1 signaling interferes with stress-coping behavior in mice[END_REF] . However, it should also be noted that some studies report no significant differences in immobility in the Porsolt FST between CB 1 R knockout and wild-type controls except when mice were exposed several times to the test [START_REF] Jacob | Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: role of glutamatergic transmission[END_REF] . Finally, mice lacking CB 1 R in cortical glutamatergic neurons showed decreased immobility in the FST, in contrast to n-3 PUFAs deficient mice, whereas mice lacking CB 1 R in GABAergic neurons display the same immobility time as littermate controls [START_REF] Steiner | Conditional cannabinoid receptor type 1 mutants reveal neuron subpopulation-specific effects on behavioral and neuroendocrine stress responses[END_REF] . Fundamental biochemical differences between CB 1 R knockouts and n-3 deficient mice may explain these behavioral discrepancies.

The selling and clinical trials of Rimonabant, a CB 1 R antagonist and anorectic antiobesity drug was suspended because of the increased risks of serious psychiatric problems including depression [START_REF] Samat | Rimonabant for the treatment of obesity[END_REF] . Thus, our data showing that in rodents, n-3 deficiency profoundly impacted on emotional behaviors might not come as a surprise.

Rather, our findings further support the clinical and epidemiological associations between n-3/n-6 imbalance and mood disorder [START_REF] Parker | Omega-3 fatty acids and mood disorders[END_REF] .

In all cases, in order to help define if dietary n-3 PUFA deficiency in rodents are responsible for behaviors underlying depression, further evaluation in a number of additional depression-related animal paradigms is required [START_REF] Krishnan | The molecular neurobiology of depression[END_REF] . Thus, whether n-3 PUFA-CB 1 R reported here could be targeted with specific dietary supplementation to improve mood disorders still remains a subject of debate [START_REF] Liperoti | Omega-3 polyunsaturated fatty acids and depression: a review of the evidence[END_REF] .

In conclusion, by linking diet to altered synaptic functions of CB 1 R in relevant brain areas, our data provide the first synaptic substrate for the impairment of emotional behavior, including depression, associated with the low levels of n-3-PUFAs frequently observed in western diets.

Methods:

Animal treatment and diet

All animal experiments were performed according to the criteria of the European Communities Council Directive (86/609/EEC) and the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals.

After mating, C57BL6/J females were fed with a diet containing 6% fat in the form of African peanut oil (rich in linoleic acid 18:2n-6) called "n-3 deficient diet" or rapeseed oil (rich in α-linolenic acid 18:3n-3) called "n-3 diet" throughout gestation and lactation [START_REF] Mingam | Uncoupling of interleukin-6 from its signalling pathway by dietary n-3-polyunsaturated fatty acid deprivation alters sickness behaviour in mice[END_REF] . After weaning, the offspring was submitted to the same diet throughout life. The mice were housed, grouped and had ad libitum food and water access. The experiments were performed in 6-16 weeks old mice.

Analysis of fatty acids in brain lipids

Lipids from total brain and PFC were extracted [START_REF] Folch | A simple method for the isolation and purification of total lipides from animal tissues[END_REF] and fatty acids were transmethylated [START_REF] Morrison | Preparation of Fatty Acid Methyl Esters and Dimethylacetals from Lipids with Boron Fluoride--Methanol[END_REF] . Fatty acid methyl esters were analyzed on a Hewlett-Packard 5890 series II gas chromatograph and a CPSil88-silica capillary column (100 m×0.25 mm internal diameter, film thickness 0.20 µm, Varian, Les Ulis, France). The injector and the detector were at 250°C and 280°C, respectively. Hydrogen was used as a carrier (inlet pressure 210 kPa). The oven was fixed 60°C for 1 min, increased to 85°C at a rate of 3°C/min and then to 190°C at a rate of 20°C/min and left at this temperature for 65 min. Fatty acid methyl esters were identified by comparison with commercial standards.

Slice preparation and electrophysiology

Whole cell patch-clamp and extracellular field recordings were made from pyramidal cells in coronal slices of mouse PrPFC and nucleus accumbens medium spiny neurons [START_REF] Lafourcade | Molecular components and functions of the endocannabinoid system in mouse prefrontal cortex[END_REF][START_REF] Mato | A single in-vivo exposure to delta 9THC blocks endocannabinoid-mediated synaptic plasticity[END_REF] . Mouse were anesthetized with isoflurane and decapitated. The brain was sliced (300 µm) in the coronal plane (Integraslice, Campden Instruments, Loughborough, UK) and maintained in physiological saline (4°C). Slices were stored The AMPA receptors (AMPAR)-mediated to NMDA receptors (NMDAR)-mediated EPSCs amplitudes ratio was measured from EPSC at +40 mV [START_REF] Kasanetz | Maturation of excitatory synaptic transmission of the rat nucleus accumbens from juvenile to adult[END_REF] . The AMPAR component of the EPSC was isolated with the NMDAR antagonist DL-AP-V (100 µM). The NMDAR-mediated EPSC was obtained by digital subtraction of the AMPAR EPSC from the dual component EPSC.

Autoradiographic studies

Mice were decapitated, brains rapidly removed and stored at -80°C. Coronal brain sections (20 μm thick) containing prelimbic and motor cortices were cut using a microtome cryostat, thaw-mounted in gelatinised slides and stored at -20°C. Ssections were then washed twice (15 min) in 50 mM Tris-HCl buffer (pH 7.4) at 4 °C, rinsed, dried and exposed to [START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF] C-sensitive films (Kodak BioMax MR, Sigma, Spain) with [START_REF] Nestler | The mesolimbic dopamine reward circuit in depression[END_REF] C-polymer standards (Amersham, Switzerland, 2 days, 4 °C).

Western blot

PFC were homogenized in lysis buffer and protein concentration determined using a BCA assay kit (Uptima, Montlucon, France). Equal amount of proteins (60 µg) were loaded on SDS/PAGE gels (10%) and transferred onto PVDF membranes (Millipore, Billerico, MA, USA). Membranes were incubated overnight (4ºC) with anti-CB 1 R (1/1000, kindly provided Dr. K. Mackie, University of Bloomington, Indiana, USA) or anti-actin antibodies (1/2500, Sigma). After washing, membranes were incubated 1 h with peroxidase-conjugated secondary anti-rabbit (1/5000, Jackson ImmunoResearch, Westgrove, PA, USA). Between each revelation, membranes were incubated (10 min, 70ºC) in stripping buffer (0.065 M Tris, 1%, SDS, 0.7% βmercapto-ethanol, pH 6.7) to remove the previous antibody. Staining was revealed with ECL-Plus Western blotting system (Perkin Elmer, Forest City, CA).

Chemiluminescence was captured and quantified by Gene Tools (Syngene).

Purification and quantification of anandamide and 2-arachidonoylglycerol

These experiments were performed as described previously [START_REF] Di Marzo | Leptin-regulated endocannabinoids are involved in maintaining food intake[END_REF][START_REF] De Marchi | Endocannabinoid signalling in the blood of patients with schizophrenia[END_REF][START_REF] Matias | Regulation, function, and dysregulation of endocannabinoids in models of adipose and beta-pancreatic cells and in obesity and hyperglycemia[END_REF] . Tissues were homogenized and extracted with chloroform/methanol/Tris-HCl 50 mM, pH 7.5

(2:1:1, v/v) containing internal deuterated standards (AEA-d 4 and 2-AG-d 5 ). The dried lipid extract was pre-purified by open bed chromatography on silica columns eluted with increasing concentrations of methanol in chloroform. Fractions for AEA and 2-AG measurement were obtained by eluting the column with 9:1 (by vol.) chloroform/methanol and were concentrated on an N2 stream evaporator. Samples were subjected to isotope-dilution liquid chromatography-chemical ionizationtandem mass spectrometric analysis. Tandem mass spectrometry, in the form of LC-MS-MS, has been used to identify AEA but also 2-AG in the picomol/gram range in both human and rat brain regions [START_REF] Richardson | Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry[END_REF][START_REF] Kingsley | LC-MS-MS analysis of neutral eicosanoids[END_REF] . Mass spectral analyses were performed on a TSQ Quantum triple quadrupole instrument (Thermo-Finnigan) equipped with an APCI source (atmospheric pressure chemical ionization) and operating in positive ion mode. The Quantum triple quadrupole instrument was used in conjunction with a Surveyor LC Pump Plus (Supelco C18 Discovery Analytical column equipped with a Phenomenex Analytical Guard Cartridge System) and cooled autosampler. The amounts of AEA and 2-AG are expressed as pmol or fmol per mg of weight tissue.

Behavioral tests

Mice were regularly handled and weighed before and during behavioral experiments.

All tests were conducted in a sound-attenuated separate experimental room, during the last four hours of the light period to avoid low spontaneous activity. Behavioral sessions were video recorded and analyzed using The Observer (Noldus, Netherlands).

Porsolt forced swim test (FST) [START_REF] Porsolt | Rodent models of depression: forced swimming and tail suspension behavioral despair tests in rats and mice[END_REF] .

Mouse were individually placed into a dark gray polyvinylchloride cylinder (15 cm diameter, 30 cm high) half-filled with water (25±1°C), so that it would neither reach the base nor the edge of the cylinder. The water was changed between subjects. Time spent immobile during the two minutes of the test was used as an index of despair-like behavior. Climbing and swimming were also measured. A mouse was considered immobile when it made only minimal movements to keep its head above water. Imipramine (Sigma, St Louis, MO, USA) was dissolved in saline (0.9%NaCI, 20 mg/kg) and administered i.p. in 0.1 mI/10 g of mouse 30 min priors to experiment.

Open field test

The apparatus consisted of a white Plexiglas-covered sawdust bedding (40 x 40 cm) with 16 cm high walls. The floor was divided into 16 green lines squares (10 x 10 cm). Each mouse was placed facing a corner and allowed to explore freely for 10 min. A video tracking system (SMART, Panlab) recorded the exact track of each mouse as well distance (cm), time spent in the inner region (central squares) and the outer region (12 squares along the perimeter of the floor), the number of outer and inner crossing and the total distance traveled. At trial end, mice were returned to their home cages, and test boxes cleaned. The percentage of time spent in the center and the time spent along the walls normalized to the total time spent in the apparatus (thigmotaxis) and total distance (in cm) were measured. All tests were conducted during the light period of the light -dark cycle.

Social investigation

Group-housed experimental subjects (4/cage) were separated and transferred to a new cage (40 x 40 cm) with fresh bedding material before the experiment. A social exploration session comprised 5 min exposure of an adult conspecific of the same age and same sex enclosed in a wire mesh cage placed in the corner of the cage [START_REF] Berton | Essential role of BDNF in the mesolimbic dopamine pathway in social defeat stress[END_REF] .

Active investigatory behavior (mainly sniffing the anogenital region, mouth, ears, trunk and tail of the adult), anxiety behavior (scraping bedding) and locomotor activity (distance in cm) were recorded.

Statistics

for 30 min at 32 -

 32 35°C in artificial cerebrospinal fluid (ACSF) containing (mM): 126 NaCl, 2.5 KCl, 2.4 MgCl 2 , 1.2 CaCl 2 , 18 NaHCO 3 , 1.2 NaH 2 PO 4 , and 11 Glucose, equilibrated with 95% O 2 /5% CO 2. Slices were stored at room temperature until recording. For recording, slices were superfused (2 ml/min) with ACSF. All experiments were done at 32-35°C. The ACSF contained picrotoxin (100 µM) to block GABA A receptors. Drugs were added at the final concentration to the ACSF. To evoke synaptic currents, 100-150 µsec stimuli were delivered at 0.1 Hz through a ACSF filled glass electrode placed either in layer II-III or in layer V-VI. There was no difference between the two sites and data were pooled together. EPSC area and amplitude were measured (graphs depict amplitudes). For extracellular field experiments, the recording pipette was filled with ACSF. The field excitatory postsynaptic potential (fEPSP) area was measured. The glutamatergic nature of the fEPSP was confirmed at the end of the experiments using the ionotropic glutamate receptor antagonist DNQX (20 µM), that specifically blocked the synaptic component (not shown). Pyramidal neurons in PrPFC layer V-VI and accumbens neurons were visualized using an infrared microscope. Whole cell patch-clamp experiments were made with electrodes containing (mM): Cesium Methane-Sulfonate (CH 3 O 3 SCs) or K + Gluconate 128, NaCl 20, MgCl 2 1, EGTA 1, CaCl 2 0.3, Na 2+ -ATP 2, Na + -GTP 0.3, Glucose 10 buffered with Hepes 10, pH 7.3, osmolarity 290 mOsm. Electrode resistance was 4-6 MOhms. If access resistance (no compensation, <25 MOhms) changed by >20%, the experiment was rejected. The potential reference of the amplifier was adjusted to zero prior to breaking into the cell. Data were recorded on an Axopatch-1D, filtered at 1-2 kHz, digitized (5 kHz, DigiData 1200), collected using Clampex 9.2 and analyzed using Clampfit 9.2 (all from Molecular Device, Sunnyvale, USA).

CB 1 R

 1 autoradiography Slides were preincubated (30 min, room temperature) in a buffer containing 50 mM Tris-HCl, 5% BSA, pH 7.4, and incubated (2 h, 37°C, same buffer) with 3 nM [ 3 H]CP55,940 (specific activity, 174.6 Ci/mmol). Non-specific binding was determined in adjacent sections by coincubation with 10 μM WIN55,212-2. Unbound radioligand was removed by washing twice (2 h ech, 4°C) in a buffer containing 50 mM Tris-HCl and 1% BSA, pH 7.4. After drying, autoradiograms were generated by apposing the tissues for 21 days at 4°C to 3 H-sensitive films (Kodak BioMax MR, Sigma, Spain). CB 1 R agonist-stimulated [ 35 S]GTPγS autoradiography Sections were preincubated (30 min, room temperature) in a buffer containing 50 mM Tris-HCl, 0.2 mM EGTA, 3 mM MgCl 2 , 100 mM NaCl, 1 mM dl-dithiothreitol and 2 mM GDP, pH 7.7, to eliminate endogenous ligands. Sections were incubated for 120 min in the same buffer containing 0.5% BSA, 3 mU/ml adenosine deaminase and 0.04 nM [ 35 S]GTPγS. Consecutive sections were incubated with 1 μM of the cannabinoid receptor agonist WIN55,212-2. Non-specific binding was determined in the presence of 10 μM guanosine-5-O-(3-thio) triphosphate (GTPγS).

  All values are given as mean ± S.E.M. Results obtained in the FST test and the open-field with WIN were analyzed by a two-way analysis of variance (ANOVA, Diet x Treatment) followed by the appropriate post-hoc test (Bonferroni). Nonpaired Student's t test was used to analyze results obtained in the social investigation and open-field tests. For electrophysiological experiments, n corresponds to the number of individual cells/slices analyzed, with at least 5 animals included in each condition. Statistical significance between groups was tested using one-way ANOVA or the Mann-Whitney test, as corresponding. In [ 35 S]GTPγSautoradiographic experiments the effect of the cannabinoid agonist was expressed as percentage of stimulation over basal activity (% = agonist binding x 100)/basal binding). For autoradiographic data, serial coronal sections were bilaterally examined between levels 2.4 and 2.0 according to a mouse brain atlas. Densitometry measurements of external (I-IV) and internal (V-VI) layers within prelimbic and motor cortices was carried our using NIH Image J software. Statistical comparison of experimental groups was made using a non-paired Student's t test, where n corresponds to the number of animals analyzed. All statistical tests were performed with GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA) using a critical probability of p < 0.05 (*p < 0.05, **p < 0.005).

Figure legends Figure 1 : 6 ( 3 ( 6 ( 1 . 6 ( 9 .

 legends1636169 Figure legends

Figure 2 :

 2 Figure 2: Endocannabinoid-dependent synaptic plasticity is absent in n-3 deficient mice. (A) In n-3 diet mice, stimulation of deep layer PrPFC synapses (arrow: 10 min at 10 Hz) induced normal eCB-mediated long-term depression (LTD, blue circles, n=15). In contrast, LTD was completely ablated in n-3 deficient mice (red squares, n=12). (B) Representative current traces showing normal LTD in a voltage-clamped (-70 mV) PrPFC layer V-VI pyramidal neuron from a mouse fed with the n-3 balanced diet (top, blue traces), and impaired plasticity in a cell from a mouse fed with the n-3 deficient diet (bottom, red traces). (C) eCB-mediated LTD was

Figure 3 :

 3 Figure 3: Nutritional n-3 deficiency did not alter other forms of synaptic plasticity (A) n-3 deficiency did neither enhanced nor depressed basal synaptic efficacy: the AMPAR/NMDAR ratio was similar in both groups (n-3 diet, blue circles: n=8; n-3deficient, red squares: n=13). (B) Bath application of the mGluR 2/3 specific agonist LY379268 (100 nM) triggered similar LTD in both groups (n-3 diet, blue circles: n=7; n-3-deficient, red squares: n=8). Error bars represent s.e.m.

Figure 4 :

 4 Figure 4: Nutritional n-3 deficiency reduces synaptic CB 1 R efficiency and CB 1 R coupling to G i/o -proteins. (A) Concentration-response curve for CB 1 R-dependent inhibition of synaptic transmission in PFC slices from n-3 and n-3 deficient mice. There was a marked reduction in the ability of the cannabinoid agonist CP55,940 to inhibit EPSC size in the PrPFC of n-3 deficient mice p < 0.05. (B) n-3 deficiency uncoupled CB 1 R from G i/o proteins, in PrPFC layer V-VI. The efficacy of the cannabinoid agonist WIN55,212-2 (1 µM) to stimulate [ 35 S]GTPγS binding was reduced in the n-3 deficient group (n-3 diet: n=5; n-3-deficient: n=5; p < 0.05). (C) CB 1 R density in prelimbic and motor cortices, measured as the specific binding of the CB agonist [ 3 H]CP55,940, was similar in n-3 and n-3 deficient mice. (D) n-3 deficiency did not alter CB 1 R protein levels in the PFC compared with normal n-3 diet. Error bars represent s.e.m.

Figure 5 . 2 [

 52 Figure 5. Anxiogenic and prodepressant-like effects of dietary n-3 deficiency (A-B) n-3 deficient mice (n=24) spent more time immobile (A) and less time swimming (B) than n-3 diet mice (n=19). Imipramine (20 mg/kg, ip) induced antidepressant-like effects in both groups: decreased in immobility time (A) and increased time swimming (B). There was a diet effect on immobility (diet, F(1,42)=7.03, p < 0.05) and swimming time (diet, F(1,41)=10.027, p < 0.01), and a treatment effect on immobility (imipramine, F(1,42)=18.45, p < 0.001) and swimming (imipramine, F(1,41)=39.094, p < 0.001) with an interaction (diet x treatment on immobility F(1,42)=5.37, p < 0.05) and on swimming F(1,41)=7.65, p < 0.01). n-3

Acknowledgments: This work was supported by INSERM, INRA and Région Aquitaine. The authors thank O. Lassalle for helping with illustrations and Dr. P. Chavis and Dr. M. Darnaudéry for helpful discussions.

supervised the entire project and wrote the manuscript.

Competing interests statement:

The authors declare that they have no competing financial interests.

Supplementary Information Titles

Journal: Nature Neuroscience

Article Title:

Nutritional Omega-3 deficiency abolishes endocannabinoid mediated neuronal functions

Supplementary Item & Number

(add rows as necessary) Supplementary Figure 1 Dietary n-3 deficiency does not change the input/output relationship at PrPFC synapses.

Supplementary Figure 2 Reduced CB1R-mediated stimulation of [ 35 S]GTPγS binding in the PrPFC of n-3 deficient mice.

Supplementary Figure 3 Nutritional n-3 deficiency does not change eCB levels.