Dr Marianne Delville
email: marianne.delville@aphp.fr

Florence Bellier

Juliette Leon

Roman Klifa

Sabrina Lizot

Hélène Vinçon

Steicy Sobrino

Romane Thouenon

Alexandrine Armance Marchal

Romane Thouennon

Alexandrine Guarrigue

Juliette Olivré

Soëli Charbonnier

Chantal Lagresle-Peyrou

Mario Amendola

Axel Schambach

David Gross

Baptiste Lamarthée

Christophe Benoist

Julien Zuber

Isabelle André

Marina Cavazzana

Emmanuelle Six

A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome

Keywords: regulatory T cell, gene therapy, scurfy, autoimmunity, IPEX, FoxP3 Abbreviation 7-AAD 7

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

Introduction

Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome is a primary immunodeficiency caused by mutations in the gene coding for the transcription factor forkhead box P3 (FOXP3) [START_REF] Wildin | X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy[END_REF][START_REF] Bennett | The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3[END_REF] . These mutations lead to the loss of function of CD4 + CD25 + regulatory T cells (Treg), a small subset of CD4 + T cells dedicated to the control of immune response [START_REF] Fontenot | Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[END_REF][START_REF] Hori | Control of regulatory T cell development by the transcription factor Foxp3[END_REF][START_REF] Khattri | An essential role for Scurfin in CD4+CD25+ T regulatory cells[END_REF] . Patients with IPEX develop of multiple autoimmune manifestations early in life [START_REF] Barzaghi | Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: An international multicenter retrospective study[END_REF] . The current treatments for IPEX syndrome include supportive therapy, immunosuppressive therapy, and hematopoietic stem cell transplantation (HSCT). Immunosuppression is usually partially effective and is often limited by infectious complications and toxicities. At present, the only curative treatment is allogeneic HSCT.

However, the lack of HLA-compatible donors and the patients' condition result in a high mortality.

Effective alternative treatments are therefore urgently needed. From HSCT, we learned that partial donor chimerism and selective expansion of Treg are sufficient for complete remission [START_REF] Kasow | Therapeutic in vivo selection of thymicderived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX[END_REF][START_REF] Seidel | Selective engraftment of donor CD4+25high FOXP3positive T cells in IPEX syndrome after nonmyeloablative hematopoietic stem cell transplantation[END_REF][START_REF] Horino | Selective expansion of donor-derived regulatory T cells after allogeneic bone marrow transplantation in a patient with IPEX syndrome[END_REF] .

Scurfy mice present a spontaneous FoxP3 mutation. Within the first 10 days of life, these mice develop a fatal disease with organ specific auto-immunity [START_REF] Godfrey | X-linked lymphoreticular disease in the scurfy (sf) mutant mouse[END_REF] that reproduces the most severe IPEX syndrome. Various studies in scurfy mouse have demonstrated that the adoptive transfer of Treg within the first two days of life is an effective treatment [START_REF] Fontenot | Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[END_REF][START_REF] Haribhai | A Requisite Role for Induced Regulatory T cells in Tolerance Based on Expanding Antigen Receptor Diversity Dipica[END_REF] . However, no one has demonstrated that Treg transfer can cure scurfy disease. A new therapeutic strategy is required to broaden the therapeutic window and mimic the patient treatments.

Several gene therapies targeting mature T cells have been successfully developed [START_REF] Kalos | Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology[END_REF][START_REF] June | Chimeric antigen receptor therapy[END_REF] . In Ragdeficient mice, the retroviral or lentiviral transfer of Foxp3 was sufficient to convert CD4 + T cells into suppressive lymphocytes able to control an inflammatory bowel disease [START_REF] Fontenot | Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[END_REF] . Furthermore, Passerini and al.'s demonstrated that the adoptive transfer of FOXP3-transduced CD4 + T cells prevented graftversus-host disease (GvHD) in a Xenograft model [START_REF] Passerini | CD4+ T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer[END_REF] . These preclinical studies suggest that the transfer of wild-type (WT) FOXP3 gene into T cells is a promising treatment (3,14). At present, two caveats prevent the translation of these preclinical studies into the clinic: the adoptive transfer of FoxP-3 transduced CD4 + T cells was used before the onset of autoimmune manifestations, and the FOXP3transduced cells only partly reproduced the Treg's transcriptional signature and suppressive function [START_REF] Fu | A multiply redundant genetic switch "locks in" the transcriptional signature of regulatory T cells[END_REF] . The expression of FOXP3 must be stable and strong enough to provide suppressive function and a surface marker is required to avoid adverse reactions related to the injection of non-corrected Tconv.

Here, we describe development and implementation of a full preclinical strategy for transferring FOXP3 into scurfy CD4 + T cells. We demonstrated that the resulting Treg were able to rescue disease manifestations in the scurfy model after the onset of autoimmune disease. This gene therapy strategy paves the way to clinical trials with curative intent in patients with IPEX.

Material and methods

Ethics

Animal procedures were approved by the animal committee (Paris University, 03/07/2017) and the Ministry of Agriculture (APAFIS#8440-2016062309559589). The procedures were performed in accordance with the EU Directive 2010/63/EU.

Mice

The scurfy phenotype was obtained by backcrossing on a B6.129S7-Rag1 tm1Mom /J background, allowing the generation of X Sf / X Sf .Rag1 -/-females. Crossing of these females with WT C57BL/6J mice resulted in only diseased X Sf /Y.Rag1 -/+ males. These males developed a scurfy phenotype. We developed a specific scurfy disease score ranging from 0 (no disease) to 21 (most severe disease) (see Supplemental Table 1).

Wild-type Treg

Splenocytes and lymph nodes were harvested from B6LY5.1 CD45.1 mice.

Scurfy CD4 + T cells

Lymph nodes were collected from 10-day-old scurfy mice, and CD4 + T cells were separated using a murine CD4 + T cell Isolation Kit (Miltenyi Biotec, Paris, France).

Lentiviral vectors

The cDNAs for a truncated codon-optimized human ΔLNGFR as a membranous reporter and a codonoptimized human FOXP3 were cloned into a pCCL backbone. We generated eight vectors in total:

four expressing FOXP3 and the four corresponding mocks.

Two vectors had a bidirectional promoter architecture. One allowed FOXP3 expression under the control of EF1α and ΔLNGFR expression under the control of PGK promoter (LNGFRp-eFOXP3 and

LNGFRp-e, respectively), and the other allowed FOXP3 expression under the control of PKG and ΔLNGFR under the control of EFS (LNGFRe-pFOXP3 and LNGFRe-p, respectively). Two bicistronic vectors were created (using the 2A self-cleaving peptide system) to allow the co-expression of FOXP3 and ΔLNGFR (namely eLNGFR.t2a.FOXP3 and eLNGFR.t2a versus. eFOXP3.t2a.LNGFR and e.t2a.LNGFR) (Fig. 1A).

T cell transduction

T cell transduction was performed as previously describe [START_REF] Delville | A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells[END_REF] . Transduced cells were stained on day 5 post-transduction, using ΔLNGFR PE antibodies (clone ME20.4-1.H4, Miltenyi Biotec), and sorted on an SH800 system (Sony Biotechnology).

Adoptive T cell transfer

Transcriptomic analysis

On day 50, 1000 CD45.1 or LNGFR + CD4 + T cells were sorted twice from lymph nodes (using a flow cytometer (SH800, Sony Biotechnology, Weybridge, UK)) directly into 5 µl lysis buffer. Smart-seq2 libraries for ultra-low-input RNA-seq were prepared as described previously [START_REF] Picelli | Smart-seq2 for sensitive full-length transcriptome profiling in single cells[END_REF][START_REF] Picelli | Full-length RNA-seq from single cells using Smart-seq2[END_REF] . Samples were sequenced on an Illumina NextSeq500 system, using the 2 x 25 bp read option. Transcripts were quantified using the Broad Technology Labs computational pipeline (42

Results

Conversion of scurfy CD4 + T cells into Treg by FOXP3 gene transfer

Four distinct vectors were generated: two bidirectional vectors using either a phosphoglycerate (PGK)/ubiquitous elongation factor 1 alpha (EF1α) (p-e) or a short version of EF1α (EFS)/PGK (e-p)

promoter cassette driving the expression of ΔLNGFR in the reverse orientation and FOXP3 in the forward position, and two bicistronic vectors using the T2A self-cleaving peptide system to co-express LNGFR and FOXP3, either LNGFR.t2a.FOXP3 or FOXP3.t2a.LNGFR (both driven by the EF1α promoter) (Fig. 1A). The corresponding four mock vectors (driving the expression of ΔLNGFR only)

were also generated.

The titers of the bidirectional vectors were more than 10 times higher than those of the bicistronic vectors (Fig. 1B). Five days after transduction at a multiplicity of infection of 10 (Fig. 1C), 5.2 to 25.2% of the WT CD4 + T cells expressed ΔLNGFR + . Spearman's coefficient (r 2 ) for the correlation between FOXP3 and ΔLNGFR expression was 0.51, 0.54, 0.66 and 0.61 for the LNGFRp-eFOXP3, LNGFRe-pFOXP3, eLNGFR.t2a.FOXP3 and eFOXP3.t2a.LNGFR vectors, respectively. The eFOXP3.t2a.LNGFR vector was excluded from further evaluation because of its low transduction efficiency. The LNGFRe-pFOXP3 construct gave a lower mean fluorescence intensity (MFI) for FOXP3 expression and was excluded. Both the LNGFRp-eFOXP3 and eLNGFR.t2a.FOXP3 constructs were tested in scurfy CD4 + T cells. As shown in Fig. 1D, the level of transduction after 5 days was significantly higher with the bidirectional LNGFRp-eFOXP3 construct than with the bicistronic eLNGFR.t2a.FOXP3 construct for both WT CD4 + T lymphocytes and scurfy CD4 + T lymphocytes (p = 0.002 and 0.007, respectively). Importantly, the MFI of FOXP3 expression was similar for CD4 LNGFRp-eFOXP3 and CD4 eLNGFR.t2a.FOXP3 in WT cells but was significantly higher with the bidirectional LNGFRp-eFOXP3 construct, compared with the T2A construct in scurfy CD4 + T lymphocytes (p=0.04) (Fig. 1E). After sorting of transduced cell and expansion, the vector copy number (VCN) in WT CD4 + T lymphocytes ranged from 0.9 to 1.9 for the bidirectional vector and from 0.9 to 1.4 in scurfy CD4 T cells. Scurfy CD4 + T cells transduced with the T2A vector were significantly less viable, not allowing VCN quantification. Since the three criteria were met for LNGFRp-eFOXP3 and its mock counterpart LNGFRp-e, they two were selected for functional evaluation (referred to hereafter as LNGFR.FOXP3 and LNGFR).

A combination of cyclophosphamide, interleukin-2 and Treg cures scurfy syndrome

Hence, we sought to develop a new therapeutic approach to skew the immune response towards the regulatory arm by depleting activated Tconv and promoting Treg expansion.

To this end, we evaluated various immunosuppressive regimens, including temsirolimus (a prodrug for sirolimus that doubles life expectancy in scurfy mice [START_REF] Chen | Mammalian target of rapamycin activation underlies HSC defects in autoimmune disease and inflammation in mice[END_REF] , anti-CD3 antibody, and cyclophosphamide (Cy). The experimental scheme consisted in injection of the immunosuppressive drug and then the transplantation of 5x10 5 congenic WT CD45.1 CD4 + CD25 high Treg (Fig. S1A, D and G). To reproducibly evaluate the scurfy mouse phenotype, we developed a specific scurfy disease score (Supplemental Table 1). Engraftment of WT Treg was quantified in various tissues at the study endpoint. When combined with Treg, temsirolimus and anti-CD3 did not significantly curb the course of scurfy disease (Fig. S1B and1E). In contrast, a combination of Cy conditioning with Treg resulted in a significantly lower scurfy score, starting from 62 days of follow-up (Fig. S1H). Chimerism at endpoint seemed lower after Cy conditioning compared to chimerism after Te or anti-CD3 treatment (Fig. S1C, F and I). But it was evaluated at a later end-point (D77) thanks to a survival improvement with Cy treatment, while analyzed at D28 and D35 respectively for Te and anti-CD3 treatment. We thus selected Cy as immunosuppressive regimen, based on scurfy scores and survival.

The percentage of WT Treg remained low (less than 2% of CD4+ cells) in all the tissues analyzed, irrespective of the type of immunosuppressive drug used (Fig. S12C, F andI). We therefore sought to increase the engraftment of Treg by evaluate different dose levels of Cy (50, 100 or 150 mg/kg body weight (data not shown) administered intraperitoneally (i.p.) to scurfy males on day 10 of life. T cell depletion was similar for all three doses, with a nadir between 3 and 5 days after Cy injection.

However, we observed growth retardation and transient alopecia, which were correlated with the dose of Cy. We therefore selected the lowest Cy dose (i.e. 50 mg/kg) in order to reduce drug toxicity. Treg transfer was performed on day 14 in mice conditioned with a single Cy injection on day 10 (Fig. S12G).

We next sought to further tip the balance in favor of Treg by promoting their expansion. On the basis of previous reports of a beneficial effect of low-dose interleukin (IL)-2 on Treg expansion [START_REF] Serreze | Immunostimulation circumvents diabetes in NOD/Lt mice[END_REF][START_REF] Tang | Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[END_REF][START_REF] Grinberg-Bleyer | IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[END_REF] , we treated scurfy recipients with IL-2 once a day for 5 days and then once a week (Fig. 2A). With the combination of Cy conditioning, Treg, and IL-2 injection, the scurfy score started to fall on day 22 after Treg transfer. The score on day 36 was significantly lower in treated mice (1.0 ± 0.3) than in control mice (4.2 ± 0.2) (p=0.0001 in an analysis of variance (ANOVA)) (Fig. 2B). Fifty days after Treg transfer, the CD45.1 + chimerism among CD4 + T cells was 2.2% ± 0.6 in lymph nodes, 3.7% ± 1.4 in the spleen, 2.1% ± 0.5 in the blood, 3.5% ± 3.2 in the liver, and 3.3% ± 0.8 in the lung (Fig. 2C).

Lastly, the survival time of Treg transferred mice (69 days) was greater than in Cy-treated mice (39 days; p=0.0004) and furthermore than non-treated mice (PBS, 28 days; p<0.0001) (Fig. 2D).

Interestingly, treatment with Cy+IL-2 alone was associated with longer survival (median survival time: 51 days), despite the lack of FOXP3 + CD4 + T cells (p =0.03). As additional evidence of Treginduced homeostatic control of the host immune system, resident T CD4 + T cells maintained a CD62L+ naïve compartment in Treg-treated mice but not in control mice (Fig. 2E). This is the first evidence to show that delayed Treg administration in combination with Cyconditioning and low dose IL-2 can improve outcomes in scurfy mice.

Scurfy CD4 + T cells transduced with the LNGFR.FOXP3 vector rescue scurfy mice

We next used our adoptive transfer model to evaluate the ability of transduced CD4 LNGFR.FOXP3 cells to control scurfy disease. Firstly, in order to assess the dose of CD4 LNGFR.FOXP3 cells required, Cyconditioned scurfy males were injected at the age of 14 days with congenic 5x10 5 CD45.1 Treg or 5x10 5 , 7.5x10 5 or 1x10 6 scurfy CD4 LNGFR.FOXP3 cells. The results were not significant between the three different doses and WT Treg. Importantly, one mouse was sacrificed before reaching the 49 days endpoint in the 1x10e [START_REF] Barzaghi | Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: An international multicenter retrospective study[END_REF] dose group because of severe scurfy injury. Therefore, the mean score at endpoint was under-evaluated in this arm. In addition, the chimerism was higher in the 7.5x10 5 group with 4.0% in the blood compared to the 1x10 6 group and 5x10 5 group (with respectively 1.2% and 0.6%, Fig. S2B andC). For those reasons, the dose of 7.5x10 5 CD4 LNGFR.FOXP3 was selected for further evaluation. The naïve CD4 + T cell compartment (characterized by CD62L staining) was restored to a similar extent by the three doses of CD4 LNGFR.FOXP3 (Fig. S2D).

Adoptive transfer experiments with 5x10 [START_REF] Khattri | An essential role for Scurfin in CD4+CD25+ T regulatory cells[END_REF] WT Treg, 7.5x10 5 CD4 LNGFR.FOXP3 cells, 7.5x10 5 CD4 LNGFR cells and vehicle (phosphate-buffered saline (PBS)) was the assessed. All mice received Cy and IL-2.

The VCN of CD4 LNGFR.FOXP3 and CD4 LNGFR ranged from 1.2 to 1.5. Mice were carefully monitored until sacrifice on day 50. Scurfy score rose above 7 in all groups in a similar manner, up to day 27 (Fig. 3A). By day 32, the mean ± standard deviation (SD) score had risen towards the endpoint to the same extent in mice that received Cy and IL-2 alone or CD4 LNGFR cells but was significantly lower in mice treated with WT Treg and CD4 LNGFR.FOXP3 cells (6.5 ± 0.5 and 4.5 ± 0.5, respectively; p=0.007 and 0.0008; respectively). More specifically, mice having received WT Treg and CD4 LNGFR.FOXP3 cells gained weight, improved eczema on the tail and blepharitis, and recovered from Cy-induced alopecia.

In contrast, mice having received vehicle or CD4 LNGFR cells failed to thrive and presented severe eczema over the whole body (Fig. S3A).

On day 50, chimerism analysis showed that the mean percentage of CD45.1 WT Treg was 5% (1.7 -12.6%) in lymph nodes, spleen, blood, liver, and lung (Fig. 3B). In mice treated with CD4 LNGFR.FOXP3 T cells, the percentage of chimerism was slightly lower, with a mean value of 3.4% (0.2 -4.6%) in lymph nodes, spleen, blood, liver, and lung (Fig. 3C). In contrast, CD4 LNGFR T cells did not expand, and percentage of chimerism remained below 1.5% in all tissues. Importantly, human FOXP3 expression in the lymph nodes was maintained 50 days after adoptive transfer in animals treated with ΔLNGFR + CD4 + cells (Fig. S3B). The ΔLNGFR + cells were sorted from the lymph nodes, and the mean ± SD VCN was 2.3±0.8 in CD4 LNGFR.FOXP3 T cells and 1.6±0.7 in CD4 LNGFR T cells (Fig. S3C).

CD62L staining of the lymph nodes (Fig. S3D) demonstrated that a subset of naïve CD4 + T cells had been restored and maintained in mice treated with Treg and CD4 LNGFR.FOXP3 cells. The percentage of CD62L + cells was 15.7±0.6% in CD4 + T cells from scurfy mice treated with Cy and IL-2 and 78.1±2.4% in WT mice. Adoptive transfer of Treg or CD4 LNGFR.FOXP3 T cells in scurfy mice increased the percentage CD62L + cells to 44.0±6.2% and 31.1±11.8%, respectively, while adoptive transfer of CD4 LNGFR T cells maintained the level at 20.8±2.5% (Fig. S3D).

Survival was significantly extended following CD4 LNGFR.FOXP3 treatment, relative to Cy-treated scurfy mice; the median survival times were 83 and 47 days, respectively (p=0.0195). Median survival was significantly longer with WT Treg (90 days) than with Cy alone (47 days) (p<0.0001). Median survival for Treg treated mice did not differ significantly from that in CD4 LNGFR.FOXP3 -treated mice (p=0.285). Likewise, PBS-and CD4 LNGFR -treated mice did not differ significantly in their survival, with median survival times of 54.5 and 53 days, respectively (p=0.8729) (Fig. 3D). After 110 days of follow-up, the degree of chimerism in CD4 + T cells from the CD45.1 Treg and CD4 LNGFR.FOXP3 groups was lower than on day 50, with mean values of 1.5% and 1.1%, respectively (Fig. S3E). Importantly, human forkhead box P3 (hFOXP3) could still be detected in CD4 LNGFR.FOXP3 T cells -demonstrating the in vivo stability of this protein in transduced scurfy CD4 + T cells.

Overall, these results demonstrated that FOXP3 expression in scurfy CD4 + T cells recapitulated suppressor function and transduced CD4 LNGFR.FOXP3 cells were able to rescue mice from scurfy autoimmune disease.

LNGFR.FOXP3-expressing scurfy CD4 + T cells significantly recapitulate Treg transcriptomic profile

To determine whether the engineered CD4 LNGFR.FOXP3 cells mimicked bona fide Treg, we explored their transcriptomic profile 35 days after adoptive transfer in scurfy recipients. FOXP3-transduced CD4 + T cells have been analyzed before but only after in vitro culture; our present experiments were designed to reveal the effect of transducing with cells that have settled in vivo, i.e. the direct impact of FOXP3 and secondary cell adaptations in vivo. We thus purified RNA from rescued scurfy mice CD4 LNGFR.FOXP3 , CD4 LNGFR T cells and WT Treg for low-input RNAseq transcriptomic analyses.

Several important observations were made. Firstly, the RNAseq reads distinguished between the transduced human FOXP3 and the endogenous murine Foxp3 (Fig. S4A). Human FOXP3 transcripts were detected in all CD4 LNGFR.FOXP3 samples at levels (~1,000 arbitrary units) that were somewhat higher than in normal human blood Treg (typically ~150-200 in our studies; Fig. S4A). Mouse Foxp3 transcripts were also detected at slightly higher levels in CD4 LNGFR.FOXP3 cells than in CD4 LNGFR cells, although these levels were still much lower than in normal murine Treg (Fig. S4B). Secondly, a comparison of the gene expression profiles of CD4 LNGFR.FOXP3 and CD4 LNGFR cells showed that FOXP3 induced a significant shift towards the prototypic transcriptional signature of Treg 23 (Fig. 4A; p=0.005 and p=0.003 for the upregulated and downregulated signatures, respectively). The direct comparison of Treg signature genes in CD4 LNGFR.FOXP3 and Treg cells (Fig. S4C) confirmed that upregulated transcripts were generally less expressed in CD4 LNGFR.FOXP3 cells than in WT Treg (this was most clear for Foxp3 and Gpr83), and that downregulated transcripts were less expressed in WT Treg than in CD4 LNGFR.FOXP3 cells.

A focus on core Treg transcripts (unpublished data; Figure 4B) showed that their levels varied significantly in CD4 LNGFR.FOXP3 cells: some were expressed at levels similar to those seen in WT Treg (e.g. Tnfrsf9, Capg, and Ctla4), whereas others were underexpressed (e.g. Gpr83, Ikzf2, Tnfrsf1b, and Foxp3). To provide a quantitative estimate, we computed a core Treg "signature score" (Fig. 4C and Table S2), where 0 and 1 correspond to expression in Tconv and Treg cells, respectively). This score varied strongly, with some transcripts being induced to full Treg levels (Ctla4, Capg) and others less so (Gpr83, Ikzf2), giving a median [interquartile range] score of 0.41 [0.25-0.74].

More generally, we also examined the transcripts differentially expressed in CD4 LNGFR.FOXP3 cells versus CD4 LNGFR cells (those with a fold-change (FC)>2 and a nominal p<0.01). The heat map (Fig. 4D) highlighted the induction of Treg signature genes in CD4 LNGFR.FOXP3 cells and also the presence of a large gene cluster of overexpressed transcripts (relative to Treg cells). A pathway analysis showed that these overexpressed transcripts were involved in cell cycling (Fig. 4D). We hypothesize that this CD4 LNGFR.FOXP3 population must be actively cycling -perhaps in response to homeostatic control that strongly drives Treg expansion in deficient environments. Thus, an analysis of the transcriptome revealed the lasting but partial restoration of Treg identity in FOXP3-transduced scurfy cells -even in the persistently inflammatory environment. Of the various Treg adoptive transfer strategies tested here, Cy conditioning was associated with the best control of autoimmunity. Cyclophosphamide has been shown to deplete the T cell niche in mice [START_REF] Huyan | Immunosuppressive effect of cyclophosphamide on white blood cells and lymphocyte subpopulations from peripheral blood of Balb/c mice[END_REF][START_REF] Salem | Kinetics of rebounding of lymphoid and myeloid cells in mouse peripheral blood, spleen and bone marrow after treatment with cyclophosphamide[END_REF] . Moreover, Cy inhibits the function of activated T cells and thus results in a relative enrichment in Treg [START_REF] Zhang | Lymphopenia and interleukin-2 therapy alter homeostasis of CD4 + CD25 + regulatory T cells[END_REF][START_REF] Wachsmuth | Posttransplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression[END_REF] . However, even low dose levels of Cy resulted in adverse events in young mice. To tip the balance between Treg and Tconv in clinical applications, another type of conditioning would be required as anti-CD3 or anti-TCR antibodies. Those strategies would allow a selective depletion of activated T cells sparing other cells subsets and limiting side-effects. However, anti-CD3 antibody was not sufficient in our model to reach an adequate level of Tconv depletion. Furthermore, low dose IL-2 has been shown to favor Treg expansion in the context of type I diabetes [START_REF] Hulme | Central Role for Interleukin-2 in Type 1 Diabetes[END_REF] , systemic lupus erythematous [START_REF] Von Spee-Mayer | Low-dose interleukin-2 selectively corrects regulatory T cell defects in patients with systemic lupus erythematosus[END_REF] and several other autoimmune diseases [START_REF] Saadoun | Regulatory T-Cell Responses to Low-Dose Interleukin-2 in HCV-Induced Vasculitis[END_REF][START_REF] Rosenzwajg | Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial[END_REF][START_REF] Mahmoudpour | Safety of low-dose subcutaneous recombinant interleukin-2: systematic review and meta-analysis of randomized controlled trials[END_REF] . Interleukin-2 also enhances the proliferation of donor-specific Treg and promotes tolerance in allogeneic transplantation [START_REF] Koreth | Interleukin-2 and Regulatory T Cells in Graft-versus-Host Disease[END_REF][START_REF] Betts | IL-2 promotes early Treg reconstitution after allogeneic hematopoietic cell transplantation[END_REF] . The safety and biological efficacy of low-dose IL-2 as a Treg inducer in a set of 14 autoimmune diseases was assessed in the TRANSREG study [START_REF] Rosenzwajg | Immunological and clinical effects of low-dose interleukin-2 across 11 autoimmune diseases in a single, open clinical trial[END_REF] . Similarly, to this study, the initial induction in our mouse model was followed by weekly maintenance injections of IL-2. Importantly, treatment with low-dose IL-2 did not worsen auto-immunity in scurfy mice -suggesting that it is possible to maintain IL-2 treatment in patients. Moreover, we observed a trend toward an extended survival with IL-2 alone. We hypothesized treatment with IL-2 could expand a cell subset with suppressive properties even in the absence of FOXP3 expression (work in progress).

Discussion

In our model of Treg adoptive transfer, the CD4 + T cells transduced with the LNGFR.FOXP3 vector were able to rescue the scurfy disease -demonstrating the lentiviral vector's efficiency to restore regulatory function. Interestingly, scurfy CD4 + T cells transduced with LNGFR.FOXP3 vector expanded more readily than those transduced with the mock LNGFR vector. This increased sensitivity to IL-2 might have been due to a higher level of CD25 expression demonstrated by flow cytometry (not shown) and in transcriptomic data (Figure 4A-C). Moreover, these adoptively transferred Treg were stably maintained for 110 days in an inflammatory context, expressed FOXP3 in a lasting manner, and thus kept their regulatory profile. Importantly, a transcriptomic analysis demonstrated that hFOXP3 expression was associated with the induction of mFOXP3 -suggesting that the homology between human and murine FOXP3 allows the human protein to act as a transcription factor in murine CD4 + T cells.

In our assay, scurfy disease was controlled slightly better by the transfer of WT Treg than by the transfer of CD4 LNGFR.FOXP3 cells. The difference might be explained by firstly, the level of chimerism with CD4 LNGFR.FOXP3 cells was half that seen with WT Treg. Secondly, our vector expressed human FOXP3 at a lower level than murine Foxp3 in WT Treg (despite higher level compared to human WT Treg, Fig. S4); this may explain why the CD4 LNGFR.FOXP3 cells do not fully recapitulate the murine Treg's transcriptomic program. However, the proportion of cells expressing human FOXP3 in the present study was higher than previously reported [START_REF] Hill | Foxp3 Transcription-Factor-Dependent and -Independent Regulation of the Regulatory T Cell Transcriptional Signature[END_REF] . Thirdly, the engineered regulatory CD4 + T cells were collected from scurfy mice, activated in vitro for 5 days. This issue would require an optimization of manufacturing procedure to reduce cell activation and improve Treg retargeting of CD4 + T cells. Preselecting naïve T cells might result in more effective suppressor activity [START_REF] Passerini | CD4+ T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer[END_REF] . In the present study, some of the scurfy mice died (mostly from the recurrence of scurfy symptoms).

Therefore, improvement of cell manufacturing to preserve naïve or T stem cell would be interesting to improve long-term latency of transduced cells. Moreover, multiple injections of Treg, higher dose levels of IL-2 and/or more frequent injections of IL-2 or IL-2 mutein to preferentially expend suppressive cells could be interesting to improve scurfy disease control.

Our present results demonstrated the feasibility of a T-cell based gene therapy approach for restoring CD4 + T cells' suppressive properties and controlling a severe autoimmune condition. Recently, Masiuk and al. developed a hematopoietic stem/progenitor cell (HSPC)-based model of gene therapy using a lentiviral vector expressing FOXP3 under the control of its endogenous promoter [START_REF] Masiuk | Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome[END_REF] . Scurfy HSPCs were engineered using this FOXP3 lentiviral vector and transplanted into WT mice, to produce corrected CD4 + T cells. The CD4 FOXP3 cells demonstrated their ability to prevent the onset of scurfy phenotype. The total number of corrected scurfy CD4 + T cells injected in scurfy neonates was over 1.8x10 [START_REF] Kasow | Therapeutic in vivo selection of thymicderived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX[END_REF] and a VCN ranging from 3.3 to 5.8. Our genetic engineering of CD4 + T cells obtained a curative effect with a slightly lower cell dose and a lower VCN. These results demonstrated the feasibility of a HSPC gene therapy but suggest that FOXP3 expression driven by the endogenous promoter might require a higher VCN and a higher CD4 + cell dose. Gene therapy with CD4 + T cells expressing FOXP3 under the control an ubiquitous promoter and gene therapy with HSPCs expressing FOXP3 under the control of its own promoter are strategies that could be used separately or serially, depending on the patient profile. Gene editing in HSPCs might also be an alternative approach for full recapitulation of the Treg ontogeny [START_REF] Goodwin | CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells[END_REF] . However, treatment with genetically engineered CD4 + T cells would require mild conditioning and would then be applicable to severely diseased IPEX patients as a full curative therapy but also as a potential bridge therapy toward stem cell gene therapy in the more severely disease patients [START_REF] Barzaghi | Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: An international multicenter retrospective study[END_REF] .

Figures' legends

Figure 1. The efficiency of FOXP3 expression by lentiviral vectors

A. Vector maps showing the design of the four vectors and their mock counterparts within the pCCL backbone:

• two bidirectional vectors:

-LNGFRp-eFOXP3 expressing FOXP3 under the control of EF1α promoter and expressing the reporter protein ΔLNGFR under the control of PGK promoter, and its mock counterpart LNGFRp-e.

-LNGFRe-pFOXP3 expressing FOXP3 under the control of PGK promoter and expressing the reporter protein ΔLNGFR under the control of the EFS promoter, and its mock counterpart LNGFRep.

• two bicistronic vectors under the control of EF1 promoter, both based on a self-cleaving T2A sequence:

-eLNGFR.t2a.FOXP3 and its mock counterpart eLNGFR.t2a.

-

  Here, we described our development of a new Treg adoptive transfer model in the scurfy mouse. This model enables the long-term functional assessment of T-cell-based gene therapy approaches for Foxp3-deficient autoimmune disease. Using a bidirectional lentiviral vector, we showed that FOXP3 gene transfer in scurfy CD4 + T cells generated potent, stable regulatory T cells that recapitulated most of the Treg's transcriptomic profile and rescued the scurfy syndrome. Previously, the transfer of splenocytes, Treg or induced Treg in scurfy mice has been shown to prevent the development of scurfy symptoms 3,11,24 . However, there are no previous reports of rescue after symptoms have developed. Our results demonstrated that Treg transfer after the appearance of symptoms rescued scurfy symptoms. This was achieved by Cy conditioning prior to Treg injection and repeated IL-2 injections to favor Treg expansion. In this model, were observed Treg engraftment, a significantly lower clinical score, and, a three-fold longer survival time (around 100 days).

Figure 3 .

 3 Figure 3. Scurfy CD4 T cells engineered with an LNGFR.FOXP3 vector rescue scurfy mice after disease onset Male scurfy mice (X Sf /Y.Rag1 +/-on a CD45.2 background) were conditioned by an i.p. injection of 50 mg/kg of Cy on day 10 and then received either vehicle (grey circle), 5x10 5 congenic CD45.1 WT Treg (salmon square), CD4 LNGFR transduced scurfy cells (purple triangle) or 0.75x10 6 CD4 LNGFR.FOXP3transduced scurfy cells (blue triangle) on day 14. Next, 1000 IU/g IL-2 were injected i.p. once a day for 5 days and then once a week. Data from at least two independent experiments are shown.

  Figure 1

  Figure 2

  -2 + PBS Cy + IL-2 + Treg Cy + IL-2 + CD4 LNGFR.FOXP3 Cy + IL-2 + CD4 LNGFR

  Figure 3

  First, scurfy mice were treated with 2 mg/kg temsirolimus (LC Laboratories, Woburn, USA) via subcutaneous injections twice a week. An anti-CD3 Fab'2 (clone 145-2C11, BioXCell, West Lebanon, USA) was injected subcutaneous (20 µg/day) once a day for 5 days, starting on day 8 after birth.

	Cyclophosphamide (European Pharmacopoeia Reference Standard, Merck KGaA, Darmstadt,
	Germany) was injected i.p. at 50, 100, or 150 mg/kg 10 days after birth. On day 10 or day 14,
	CD4 + CD25 + CD45.1 + cells or engineered CD4 + T cells from scurfy mice were injected i.p.. In the
	indicated experiments, human IL-2 (PROLEUKIN®, Novartis, Basel, Switzerland) was injected i.p. at
	1000 IU/g once a day for 5 days and then once a week. A reference survival curve of untreated scurfy
	mice was generated from all experiments.

A specific combination of cyclophosphamide conditioning, IL-2 treatment and Treg transfer rescues scurfy syndrome

  eFOXP3.t2a.LNGFR, and its mock counterpart e.t2a.LNGFR. type transduced with LNGFR.t2a.FOXP3 (p=0.04 in a Mann-Whitney test). n=3 independent experiments for WT CD4+ T cells and n=2 independent experiments for scurfy CD4+ cells.

	LTR: long terminal repeat; WPRE6*: woodchuck hepatitis virus post-transcriptional regulatory element; PolyA unid: unidirectional poly-adenylation sequence; SIN: self-inactivating. B. Quantification of the titers of vector used for transduction. C. Representative flow cytometry dot plots showing the expression of human FOXP3 and ΔLNGFR on WT murine CD4 + T cells 5 days after transduction with all the constructs expressing FOXP3. The correlation between FOXP3 expression and ΔLNGFR expression was quantified by calculating Spearman's correlation coefficient (r 2 =0.51, 0.54, 0.66 and 0.61 for the LNGFRp-eFOXP3, LNGFRe-pFOXP3, eLNGFR.t2a.FOXP3, and eFOXP3.t2a.LNGFR vectors, respectively). D. Transduction efficacy quantified as the percentage of ΔLNGFR on day 5 after transduction in WT and scurfy CD4 + T cells. Transduction efficiency was significantly higher with the LNGFRp-eFOXP3 vector than with the LNGFR.t2a.FOXP3 vector for both WT CD4+ T cells (black circles) and scurfy CD4+ T cells (grey squares) (p=0.002 and 0.007, respectively, in a Mann-Whitney test:). In contrast, transduction efficacy with the mock vectors was higher with the T2A construct (p=0.02 and 0.04 in WT and scurfy CD4 + T cells, respectively). n=3 independent experiments for WT CD4+ T cells and n=2 independent experiments for scurfy CD4+ Tc ells. E. The geometric MFI for FOXP3 expression was quantified on day 5 post-transduction (gated on Scurfy males (X Sf /Y.Rag1 +/-on a CD45.2 background) were conditioned by an i.p. injection of 50 mg/kg cyclophosphamide on day 10 and then received 5x10 5 congenic CD45.1 WT Treg on day 14. Next, 1000 IU/g IL-2 was injected i.p. once a day for 5 days and then once a week. In an initial experiment, all mice were sacrificed on day 50 for flow cytometry analysis. Survival was analyzed in a second experiment (n=3 mice per group). line), Cy and IL-2 (Cy+IL-2+PBS; (dashed grey line) or Cy, IL-2 and 5x10 5 Treg (Cy+IL-2+Treg; pink line). There was a significant difference (p=0.0004 in a log rank test) between Cy+IL-2+PBS and Cy+IL-2+Treg groups (n=6 mice per group). E. A representative flow cytometry histogram of CD62L expression, gated on CD4+ T cells in mice that had received Treg (dark grey) or vehicle (light grey). Cy cyclophosphamide; IL: interleukin (n=3 CD4 Figure 2. A. Rescue of scurfy mice mice per group).

+ ΔLNGFR + ) in WT cells (black circles) or scurfy cells (grey squares). The hFOXP3 MFI in WT CD4 T cells was similar with LNGFRp-eFOXP3 and LNGFR.t2a.FOXP3 vectors. The MFI was significantly higher in scurfy CD4 T cells transduced with LNGFRp-eFOXP3 vector than in the same 20 cell B. The scurfy disease score was rated (as described in the Methods) in Treg-treated mice (grey squares) and vehicle (PBS)-treated mice (black triangles). Differences were apparent after day 31 (p=0.003 in a Mann-Whitney test) and after day 38 (p<0.001). Cy: cyclophosphamide; IL: interleukin. C. Flow cytometry analysis of CD45.1 chimerism, gated on CD4 + T cells in the lymph nodes, spleen, blood, liver, and lung. Cy: cyclophosphamide; IL: interleukin (n=3 mice per group). D. Survival of scurfy mice untreated (PBS, plain dark line), treated with Cy only (Cy, continuous grey

Acknowledgement

We are grateful to Aurélia Dujardin, Camila Piat, Laura Zapata, Jennifer Alonso and Emilie Panafieu from the SFR Necker animal facility, Sophie Berissi, Noémie Gadessaud and Sonita Ing from the SFR Necker histology facility, and Olivier Pellé and Corinne Cordier from the SFR Necker cytometry facility. We also thank Gisèle Froment, Didier Nègre and Caroline Costa from the lentiviral production facility/SFR BioSciences Gerland-Lyon Sud (UMS3444/US8). This work was funded by a European Research Council grant (Gene for Cure: E16145KK).

Funding acquisition Isabelle André: Conceptualization, Methodology, Writing -Review & Editing, Supervision Emmanuelle Six: Conceptualization, Methodology, Writing -Review & Editing, Supervision

Data sharing

Data have been deposited in GEO under accession number GSE166017.

Authorship Contributions

A. A volcano plot (the FC versus the p value) of the transcriptomes of CD4 LNGFR.FOXP3 vs. CD4 LNGFR cells on day 50. The Treg upregulated signature (in red), downregulated signature (in blue) [START_REF] Hill | Foxp3 Transcription-Factor-Dependent and -Independent Regulation of the Regulatory T Cell Transcriptional Signature[END_REF] and core Treg gene annotations are highlighted [START_REF] Zemmour | Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR[END_REF] . The values in the upper half represent the number of corresponding Treg signature genes induced (right) or repressed (left), with the number of upregulated signature genes in red and the number of downregulated signature genes in blue. P-values for the Treg signature enrichment were obtained in a chi-squared test. B. A heat map showing expression of the core Treg genes [START_REF] Zemmour | Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR[END_REF] . The values correspond to the FC for each gene in each sample, normalized against the mean value for CD4 LNGFR cells.


Pauline Tirelle

Jonathan Breton

Alexandre Kauffmann

Wafa Bahlouli

Clément L'huillier

Emmeline Salameh

Asma Amamou

Marine Jarbeau

Charlène Guérin

Alexis Goichon

Jean-Claude Do Rego

Pierre Déchelotte

David Ribet

Moïse Coëffier
email: moise.coeffier@univ-rouen.fr

Gut microbiota depletion affects nutritional and behavioral responses to activity-based anorexia model in a sex-dependent manner

Keywords: anorexia, gut microbiota, gut brain axis, anxiety-like behavior, activity-based anorexia

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

Introduction:

Anorexia Nervosa (AN) is an eating disorder described by the Diagnostic and Statistical Manual of Mental Disorders fifth edition (DSM-V) [START_REF] Mitchell | Diagnostic criteria for anorexia nervosa: Looking ahead to DSM-V[END_REF] and is the mental illness with the highest mortality rate [START_REF] Gravina | Medical Complications in Anorexia and Bulimia Nervosa[END_REF] , which makes it a major public health problem. AN is a multifactorial disease with female predominance, the lifespan prevalence being 1.4% for women and 0.2% for men [START_REF] Galmiche | Prevalence of eating disorders over the 2000-2018 period: a systematic literature review[END_REF] . AN is characterized by an undernutrition (body mass index (BMI) < 18.5 kg/m 2 ), an intense fear of gaining weight and disturbance in body shape perception. Psychiatric comorbidities such as anxiety and depression are frequently observed [START_REF] Junne | The relationship of body image with symptoms of depression and anxiety in patients with anorexia nervosa during outpatient psychotherapy: Results of the ANTOP study[END_REF] . AN is a multifactorial disease with biological, psychological, environmental and social influences [START_REF] Arcelus | Prevalence of eating disorders amongst dancers: a systemic review and meta-analysis[END_REF] .

Over the last decade, the role of the gut-brain axis in the regulation of eating behavior [START_REF] Van De Wouw | Microbiota-Gut-Brain Axis: Modulator of Host Metabolism and Appetite[END_REF] and anxietydepressive disorders [START_REF] Clarke | The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner[END_REF] has emerged. Indeed, the role of the gut microbiota on host satiety has been highlighted [START_REF] Breton | coli Proteins Activate Host Satiety Pathways following Nutrient-Induced Bacterial Growth[END_REF] and gut microbiota dysbiosis (abundance and diversity) has been reported in anorectic patients 9 10 . Furthermore, it has been suggested that modifications of gut microbiota in anorectic patients are associated with BMI [START_REF] Million | Correlation between body mass index and gut concentrations of Lactobacillus reuteri , Bifidobacterium animalis , Methanobrevibacter smithii and Escherichia coli[END_REF] and body composition [START_REF] Mörkl | Gut microbiota and body composition in anorexia nervosa inpatients in comparison to athletes, overweight, obese, and normal weight controls[END_REF] , and are not completely restored after refeeding [START_REF] Mack | Is the Impact of Starvation on the Gut Microbiota Specific or Unspecific to Anorexia Nervosa? A Narrative Review Based on a Systematic Literature Search[END_REF] . Interestingly, preliminary studies and case reports evaluating fecal microbiota transplantation underlined putative role of gut microbiota in the regulation of energy homeostasis during AN [START_REF] De Clercq | Weight Gain after Fecal Microbiota Transplantation in a Patient with Recurrent Underweight following Clinical Recovery from Anorexia Nervosa[END_REF][START_REF] Prochazkova | Microbiota, Microbial Metabolites, and Barrier Function in A Patient with Anorexia Nervosa after Fecal Microbiota Transplantation[END_REF][START_REF] Hata | The Gut Microbiome Derived From Anorexia Nervosa Patients Impairs Weight Gain and Behavioral Performance in Female Mice[END_REF] . Finally, the gut microbiota also affects mood disorders, particularly anxietydepressive disorders. Indeed, patients with anxiety-depressive disorders exhibited gut microbiota dysbiosis [START_REF] Winter | Gut microbiome and depression: what we know and what we need to know[END_REF] and fecal microbiota transplantation from depressive patients to germ-free animals was associate with the occurrence of altered behaviors [START_REF] Kelly | Transferring the blues: Depression-associated gut microbiota induces neurobehavioural changes in the rat[END_REF] . In addition, Neufeld et al reported reduced anxiety-like behavior and altered neurochemical pathways in amygdala and hippocampus in germfree mice [START_REF] Neufeld | Reduced anxiety-like behavior and central neurochemical change in germ-free mice[END_REF] .

Among animal models used to decipher the pathophysiology of eating disorders and particularly to mimic AN, the activity-based anorexia (ABA) model is one of the most commonly used [START_REF] Schalla | Activity Based Anorexia as an Animal Model for Anorexia Nervosa-A Systematic Review[END_REF] . It combines a free access to a running wheel and a progressive food access limitation. It leads to a severe body weight loss, an early physical hyperactivity, and alterations in brain structures involved in eating behavior [START_REF] Nobis | Alterations of proteome, mitochondrial dynamic and autophagy in the hypothalamus during activity-based anorexia[END_REF][START_REF] Scharner | Activity-based anorexia activates nesfatin-1 immunoreactive neurons in distinct brain nuclei of female rats[END_REF] and anxiety disorders [START_REF] Scharner | Activity-based anorexia activates CRF immunoreactive neurons in female rats[END_REF][START_REF] Kinzig | Adolescent activity-based anorexia increases anxiety-like behavior in adulthood[END_REF] . In addition, ABA mice exhibited inflammation [START_REF] Belmonte | A role for intestinal TLR4-driven inflammatory response during activity-based anorexia[END_REF] and an intestinal hyperpermeability [START_REF] Jésus | Alteration of intestinal barrier function during activity-based anorexia in mice[END_REF] . Recently, we characterized the gut microbiota alteration occurring in ABA mice and observed similarities with the dysbiosis observed in AN patients [START_REF] Breton | Gut microbiota alteration in a mouse model of Anorexia Nervosa[END_REF] . The physiological consequences of these gut microbiota alterations during ABA remain however unknown. Thus, we aimed to characterize the effects of gut microbiota depletion during the ABA model. As differential responses were observed in the ABA model according to the sex [START_REF] Achamrah | Sex differences in response to activity-based anorexia model in C57Bl/6 mice[END_REF] , we included both male and female mice in our study. As gut microbiota contributes to the intestinal immune system maturation [START_REF] Hapfelmeier | Reversible Microbial Colonization of Germ-Free Mice Reveals the Dynamics of IgA Immune Responses[END_REF] , gut microbiota depletion was achieved by antibiotics treatments [START_REF] Tirelle | Comparison of different modes of antibiotic delivery on gut microbiota depletion efficiency and body composition in mouse. under review[END_REF] . We then monitored body weight, food intake, physical activity and evaluated body composition, anxiety-like behavior, central neuropeptide response and intestinal parameters such as intestinal permeability.

Material and Methods:

Animals

Animal experiment was approved by the regional ethical committed CENOMEXA (authorization N/05-11-12/28/11-15). Experiments were carried out in accordance with current French and European regulations. Seven weeks old male and female C57BL/6Jrj mice (Janvier Labs, Le-Genest-Saint-Isle, France) were acclimated and housed at 23°C±2°C on reverse cycle (dark phase from 10:00 am to 10:00 pm), with water and food free-access (n=4 / cage). Animals were assigned randomly to various experimental groups.

Antifungal-antibiotics treatment

To deplete gut microbiota, mice received a cocktail of antifungal and antibiotics (ATB) in water until the end of the experiment by oral gavages (10µL/g of body weight) twice a day (d) as previously described [START_REF] Tirelle | Comparison of different modes of antibiotic delivery on gut microbiota depletion efficiency and body composition in mouse. under review[END_REF] . Briefly, from d1 to d3, only antifungal treatments were performed by supplying 0.1mg/mL amphotericin-B (Sigma-Aldrich, Saint-Louis, MO, USA). Then, an antibiotic mixture (Vancomycin 5 mg/mL, Neomycin 10 mg/mL, Metronidazole 10 mg/mL, Ampicillin 10 mg/mL) completed the treatment until the end of the protocol. Untreated mice received water by gavages.

Activity-based anorexia (ABA) model

At the beginning of the ABA procedure (d10), mice were placed individually in standard cage (CT and CT+ATB groups) or cages with an activity wheel (ABA and ABA+ATB groups). The ABA model has been performed as previously described [START_REF] Jésus | Alteration of intestinal barrier function during activity-based anorexia in mice[END_REF] . ABA model combines a free running wheel access and a progressively limited food access from 6h/day to 3h/day (Supplemental Fig. S1). Food was given at the beginning of dark phase (10:00 am). Animals had free access to water. Running wheel activity has been continuously recorded with Activity Wheel software (Intellibio, Seichamps, France). Moreover, food intake and body weight were daily measured. If body weight loss was higher than 20% on 3 consecutively days, animals were euthanized in accordance with the ethical procedure.

Body composition

At d13 (before the limitation of food access) and d27 (end of the protocol), body composition was assessed on vigil animals using Minispec LF110 (Brucker, Wissembourg, France), a fast-nuclear magnetic resonance method (n= 8/6 for male and female).

Open field test

In order to assess anxiety-like behavior, Open Field tests were performed at d13 and d27 after the feeding period (at 01:00 pm) (n= 8/6 for male and female). Data were collected with Fusion Software (Omni-Tech Electronics, Eckington, UK).

Quantification of faecal bacterial density flow cytometry

The efficiency of gut microbiota depletion was assessed by quantifying the decrease of bacterial density in mouse faeces using flow cytometry, as previously described [START_REF] Tirelle | Comparison of different modes of antibiotic delivery on gut microbiota depletion efficiency and body composition in mouse. under review[END_REF] . Briefly, faecal suspensions were stained with Syto TM BC (Molecular Probes, Eugene, OR, USA) and fixed in 1X PBS-0.5% paraformaldehyde before being analysed by flow cytometry [START_REF] Tirelle | Comparison of different modes of antibiotic delivery on gut microbiota depletion efficiency and body composition in mouse. under review[END_REF] .

Euthanasia and sample collection

Mice were anesthetized by intraperitoneal injection of Ketamine/Xylazine solution (40 and 1 mg/kg of body weight, respectively). Blood was sampled by puncture in the portal vein and plasma samples were then collected after centrifugation (3000 g; 20 min; 4°c). Brain was removed in order to isolate hypothalamus and hippocampus. Colon was collected and immediately washed with ice-cold PBS, 1 cm-long sections were then performed and frozen in liquid nitrogen. All samples were stored at -80°C until analysis.

Intestinal permeability assessment

Global intestinal permeability was assessed by administration of a solution of fluorescein isothiocyanate-Dextran (FITC-Dextran 4 kDa) at 40 mg/mL by oral gavage (10 µL/g of body weight), 3 hours before euthanasia, as previously described [START_REF] Ghouzali | Targeting immunoproteasome and glutamine supplementation prevent intestinal hyperpermeability[END_REF] . Then, FITC-Dextran level was then measured on plasma samples using a spectrofluorometer (Chameleon Lablogic, Sheffield, UK; excitation = 485 nm; emission = 535 nm; Mikro Win 2000 Software).

Evaluation of plasma leptin, adiponectin and corticosterone

Plasma leptin and adiponectin levels were assessed using MILLIPLEX assay (Merckmillipore, Fontenay sous Bois, France) or enzyme-like immunosorbent assay (Invitrogen, Carlsbad, CA, USA), both according to the manufacturer's instructions. Plasma corticosterone levels were measured by a Corticosterone ELISA kit (Abnova, Ann Arbor, MI, USA) according to the manufacturer's instructions.

RNA extraction and RT-qPCR

Total hypothalamus, hippocampus and colonic RNAs were extracted by Trizol method (Invitrogen, Carlsbad, CA, USA) following manufacturer's guidelines. After DNAse treatment (Promega, Charbonnières-les-Bains, France), RNAs were reverse transcribed as previously reported 8 . Then, qPCR was performed using SYBRGreen technology on a Bio-Rad CFX96 real-time PCR system (Bio-Rad Laboratories, Marnes la Coquette, France). RPS18 gene was used as a housekeeping gene. Specific primer sequences of targeted mouse genes are displayed in Table 1. The values were obtained by the conversion of cycle threshold on concentration value by using a standard curve.

Statistical analysis

All data were analysed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as mean ± standard error to mean. Shapiro test was used to assess distribution parameters. Data were analysed by repeated two-way ANOVA (time x group) or two-way ANOVA (ABA x ATB) followed by Bonferroni posthoc tests, as appropriate. Results were considered significant when p-value was lower than 0.05.

Results:

3.1. Impact of gut microbiota depletion during activity-based anorexia in male mice.

In order to control the efficiency of the ATB-mediated gut microbiota depletion, we compared bacteria abundance in faeces obtained before and after 15 days of ATB treatment. We observed a strong decrease in faecal bacterial density at day 15 (98% of depletion, Fig. 1), indicating that the gut microbiota is efficiently depleted before starting the ABA procedure.

At d13 (i.e. before the limitation of food access), we observed that gut microbiota depletion did not induce body weight changes in any groups (Supplemental Fig. S2A). Mice with access to an activity wheel exhibited a decreased fat mass (Supplemental Fig. S1B). Furthermore, ATB treatment induced an increase of fat mass (Supplemental Fig. S2B) and a decrease of lean mass (Supplemental Fig. S2C). We also evaluated mice behavior and did not observe major effects of ATB treatment (Supplemental Fig. S2D-I), which was only associated with a reduction of the distance travelled at the centre of open-field system (Supplemental Fig. S2E).

Then, we started to decrease food access and followed-up mice until day 28. In CT mice, ATB treatment affected neither food intake nor body weight until day 28. As previously reported [START_REF] Jésus | Alteration of intestinal barrier function during activity-based anorexia in mice[END_REF] , ABA mice exhibited a decrease of food intake (data not shown), which is associated to a marked decrease of body weight at day 28 (15.2% compared to day 16, Fig. 2A,B). Interestingly, the body weight loss was less pronounced in ATB-treated ABA mice (10% compared to day 16) compared to untreated ABA mice (Fig. 2A,B), while food intake remained unchanged (data not shown). In response to ABA model, mice exhibited a modification of body composition with a reduction of both fat (-27%) and lean masses (-20%, Fig. 2C,D). Accordingly, plasma leptin markedly decreased in ABA mice (Fig. 3A), while adiponectin remained unaffected (Fig. 3B). In ATB-treated mice, we observed higher fat mass and lower lean mass, as already observed before the limitation of food access, both in CT and ABA.

Indeed, ATB-treated CT mice showed an increase of fat mass of approximately 60% and a decrease of lean mass of 15% compared to untreated CT mice, whereas ATB-treated ABA mice exhibited an increase of 79% in fat mass and a decrease of 16% in lean mass compared to untreated ABA mice (Fig. 2C,D). ATB did not affect plasma leptin neither in CT nor in ABA mice (Fig. 3A), but increased plasma adiponectin level (ANOVA p (ATB) < 0.05, Fig 3B). In accordance with the absence of modification in food intake in ATB-treated ABA mice, hypothalamic NPY and POMC mRNA levels were not modified by ATB treatment in ABA mice (Fig. 4 A,B). Nevertheless, the increase of anticipatory physical activity was less pronounced in ATB-treated ABA mice compared to untreated ABA mice (Fig. 2E).

As we and others previously reported that intestinal permeability is affected either during ABA model [START_REF] Jésus | Alteration of intestinal barrier function during activity-based anorexia in mice[END_REF] or in response to ATB treatment [START_REF] Feng | Antibiotics induced intestinal tight junction barrier dysfunction is associated with microbiota dysbiosis, activated NLRP3 inflammasome and autophagy[END_REF] , we measured intestinal permeability in our model.

We observed that intestinal permeability was not significantly modified in male mice in our tested conditions (Fig. 3C).

We then focused on anxious behavior that was assessed at day 27. ABA mice exhibited altered behavior compared to CT mice: the distances travelled by mice at the center and at the periphery of the open field area were decreased while the time spent in these two areas remained unchanged. Consequently, the ratio distance / time decreased both at the center and at the periphery (Fig. 5 A-E). In addition, immobility time was increased in ABA mice compared to CT mice (Fig. 5G). ATB-induced gut microbiota depletion was associated with a more pronounced decrease of the travelled distance and of the ratio distance / time at the periphery in ABA mice compared to untreated ABA mice (Fig. 5E,F). A similar trend was observed at the center but difference did not reach significance (Fig. 5B). Interestingly, this effect was not observed in ATB-treated CT mice. In addition, ATB-treated ABA mice exhibited a reduced vertical activity compared to other groups (Fig. 5H). Plasma corticosterone remained unchanged (data not shown), while the increase of hypothalamic CRH mRNA observed in untreated ABA mice was totally blunted by ATB treatment (Fig. 5I). Hypothalamic TNFα and TLR4 mRNA levels were not modified (data not shown). In the hippocampus, we evaluated dopaminergic and serotoninergic systems by assessing dopamine and serotonin receptors. For DRD1, DRD2 and 5-HT1A (Htr1a gene), the ANOVA p-value for interaction was significant while there was no difference for 5-HT4 (Htr4 gene, Fig. 6A-D). Our data suggest that ATB treatment blunted the increase of DRD1 and DRD2 mRNA in ABA mice compared to CT (Fig. 6A,B). A similar pattern was also observed for TNFα mRNA (Fig. 6E) By contrast, 5-HT1A mRNA level was only decreased in ATB-treated ABA mice compared to ATB-treated CT mice but not in untreated ABA mice (Fig. 6C). Finally, BDNF mRNA levels remained unchanged (Fig. 6F).

Impact of gut microbiota depletion during activity-based anorexia in female mice.

As the prevalence of anorexia nervosa is higher in females than in males 34 and as mice exhibit sexdependent differential responses to the ABA model [START_REF] Achamrah | Sex differences in response to activity-based anorexia model in C57Bl/6 mice[END_REF] , we thus decided to perform similar experiments in female mice.

As observed in males, ATB treatment led to a marked depletion of bacteria in mouse faeces (95% of depletion, Supplemental Fig. S3) with no impact on body weight but an increase of fat mass and a reduction of lean mass (Supplemental Fig. S4A-C). ATB treatment did not markedly affect female behavior, as we only observed a decrease of the time spent at the center of the open field area without any difference for the other measured parameters (Supplemental Fig. S4D-I).

After the beginning of the food access limitation, ABA female mice exhibited a body weight loss until day 22 (-21.7% vs day 16), but then partially recovered at day 28 (-11.1% vs day 16, Fig. 7A), as previously reported [START_REF] Nobis | Alterations of proteome, mitochondrial dynamic and autophagy in the hypothalamus during activity-based anorexia[END_REF] . ATB-treated ABA mice exhibited a limitation of the body weight loss until day 22 compared to untreated ABA mice (-12.1% vs day 16, p<0.05, Fig. 7A). However, at day 28, body weight change was not different between ATB-treated and untreated ABA female mice (Fig. 7B). As observed in male mice, ATB affected body composition with an increase of fat mass and a decrease of lean mass in female mice submitted to the ABA model (Fig. 7C,D). However, plasma leptin and adiponectin concentrations were lower in ATB-treated ABA mice compared to untreated ABA mice (Fig. 8A,B). Food intake was reduced in ABA mice compared to CT mice, but was not affected by ATB treatment (data not shown) that was in agreement with the observed hypothalamic mRNA levels for NPY and POMC (Supplemental Fig. S5). In contrast to male mice, ATB-treated ABA female mice developed a similar anticipatory physical activity compared to untreated ABA mice (Fig. 7E). We did not observe any major effects of ATB treatment on female behavior at the end of the ABA protocol. Indeed, although ABA protocol induced a decrease of the distance travelled at the periphery of the open field area, an increase of immobility time and a decrease of vertical activity, ATB-treated ABA female mice did not exhibit exacerbated or blunted behavior compared to untreated ABA mice (Fig. 9 A-G). Accordingly, ATB treatment affected neither plasma corticosterone (data not shown) nor hypothalamic CRH mRNA expression (Fig. 9I) nor hippocampal dopaminergic and serotoninergic receptors mRNA levels (Supplemental Fig. S6). Finally, ATB-treated ABA female showed a marked increase of intestinal permeability compared to untreated ABA mice and ATBtreated CT mice (Fig. 8C) that was not observed in male mice.

Discussion:

Recent data suggest that the gut microbiota contributes to the pathophysiology of eating disorders and particularly of AN [START_REF] Breton | Intestinal microbiota and Anorexia Nervosa[END_REF] . Gut dysbiosis has been described in AN patients before and after refeeding 35 . These data have also been observed in ABA model both in rats 36 and mice [START_REF] Breton | Gut microbiota alteration in a mouse model of Anorexia Nervosa[END_REF] . Indeed, in a recent study, we characterized the gut microbiota alterations occurring in ABA mice [START_REF] Breton | Gut microbiota alteration in a mouse model of Anorexia Nervosa[END_REF] and observed some similarities with the dysbiosis reported in AN patients. We thus aimed to evaluate the contribution of gut microbiota in the ABA model, by monitoring the consequences of antibioticsinduced depletion. We report that antibiotics-induced gut microbiota depletion induces a similar modification of body composition in female and male mice, is associated with a limitation of body weight loss in the activity-based anorexia model in both sexes, but triggers a sex-dependent alteration of anxiety-like behavior.

Before the progressively time-limited food access, voluntary activity induced a sex-dependent modification in body composition of untreated mice. In contrast, antibiotics-induced gut microbiota depletion similarly induced an increase of adiposity and a reduction of lean mass in both sexes. Of note, body weight remained unaffected by antibiotics in accordance with previous data showing that different antibiotics cocktails, did not affect body weight both in female 37 and male 38, 39 mice and Siberian hamsters 40 .

By contrast, after the beginning of the food access limitation, mice with depleted microbiota exhibit a less pronounced body weight loss compared to untreated mice. This effect was observed in both female and male mice, even if it seems to be more pronounced in males. As observed before the limited food access, antibiotics treatment was associated with an increase of fat mass and a decrease of lean mass in response to the ABA mocel whatever the sex. To our knowledge, our study shows for the first time that gut microbiota depletion induced by antibiotics limits body weight loss during ABA model in both female and male mice. Contradictory data have been shown in germ-free mice that are less able to survive to starvation 41 . In the present study, we did not observe modification of food intake between untreated and antibiotics-treated mice, also during ABA model.

Concerning body composition, antibiotics treatment was associated with a reduction of muscle mass in male mice 38 . Similar data have been observed in germ-free male mice 42 . However, in those studies, the authors did not report on the amount of fat mass. By contrast, in female mice, antibiotics in early life did not affect lean mass but increased adiposity 37 . However, to our knowledge, the comparison of the effects of gut microbiota depletion on body composition between female and male mice remain until now undocumented. In Siberian hamsters, Sylvia et al studied the sex-specific modulation of microbiota in body weight, but did not assess body composition 40 . As previously reported [START_REF] Achamrah | Sex differences in response to activity-based anorexia model in C57Bl/6 mice[END_REF] , female and male mice exhibit different responses during ABA model. Indeed, in females, body weight loss was maximal at day 22, and then mice recovered that contributes to hide the differences between antibiotics-treated and untreated mice, and may explain the increased adiposity in female ABA mice compared to controls. By contrast, in males, the body weight loss persists until the end of protocol. In the present study, we thus demonstrated that antibiotics treatment increased adiposity and reduced lean mass in young adult mice in a sex-independent manner.

Anxiety and depression are frequent comorbidities during anorexia nervosa 43,44 and the role of microbiota-gut-brain axis in these comorbidities has been suggested 45 . Interestingly, mice receiving the transplantation of gut microbiota from patients with anxiety exhibit anxious behavior [START_REF] De Palma | Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice[END_REF] , suggesting that the microbiota can be involved in psychiatric comorbidities in anorectic patients. In our study, before the limitation of food access, antibiotics treatment was not associated with major modification of behavior of female and male mice placed in standard cages or in cages equipped with an activity wheel. We only observed a decrease of the distance travelled at the center of the open field area in male mice. At the end of ABA model, only male mice showed an altered behavior in response to antibiotics treatment. Indeed, the distance travelled at the periphery of the open field area was reduced in antibiotics-treated male mice. We observed a statistical antibiotics effect (2-way ANOVA) suggesting an increased immobility time, which is in consistent with the observed decrease of vertical activity. Along the same line, anticipatory physical activity was reduced in antibioticstreated male mice compared to untreated mice. Finally, antibiotics treatment completely blunted the increase of hypothalamic CRH mRNA expression in response to ABA model. These alterations were not all present in female mice. Previous data focusing on broad-spectrum antibiotics showed that long-term exposure to ceftriaxone reduced both anxiety and aggressive behavior in male BALB/c mice [START_REF] Zhao | Long-Term Exposure to Ceftriaxone Sodium Induces Alteration of Gut Microbiota Accompanied by Abnormal Behaviors in Mice[END_REF] . By contrast, ampicillin administration increased anxiety and immobility in male BALB/c mice 39 . In Siberian hamsters, aggressive behavior was reduced by enrofloxacin in both females and males but only male animals restored it to control level after stopping treatment 40 . In humans, antibiotics exposure led in contrast to an increased risk to develop anxiety-like behaviors [START_REF] Lurie | Antibiotic exposure and the risk for depression, anxiety, or psychosis: a nested case-control study[END_REF] . These behavioral changes were associated with gut microbiota alterations 40,[START_REF] Zhao | Long-Term Exposure to Ceftriaxone Sodium Induces Alteration of Gut Microbiota Accompanied by Abnormal Behaviors in Mice[END_REF]39 . Interestingly, anxiety-like behavior has also been studied in germ-free animals. The absence of microbiota was associated with an anxiolytic behavior both in female [START_REF] Neufeld | Reduced anxiety-like behavior and central neurochemical change in germ-free mice[END_REF] and male [START_REF] Lurie | Antibiotic exposure and the risk for depression, anxiety, or psychosis: a nested case-control study[END_REF] germ-free mice. To our knowledge, we showed here for the first time that gut microbiota depletion induced by antibiotics alters the behavioral response to ABA model but only in male mice. Previous data reported gut microbiota dysbiosis in response to ABA 36,[START_REF] Breton | Gut microbiota alteration in a mouse model of Anorexia Nervosa[END_REF][START_REF] Trinh | Gut microbiota and brain alterations in a translational anorexia nervosa rat model[END_REF] but it is interesting to note that these data have been obtained in male 36 and female [START_REF] Trinh | Gut microbiota and brain alterations in a translational anorexia nervosa rat model[END_REF] rats and in male mice [START_REF] Breton | Gut microbiota alteration in a mouse model of Anorexia Nervosa[END_REF] . It may be interesting to further compare gut microbiota alterations between male and female mice in response to ABA model. The underlying mechanisms or microbiota metabolites involved in the microbiota-gut-brain axis remain to be studied during the ABA model.

Faecal and plasma metabolome has recently been described in response to the ABA model, but only in female mice [START_REF] Breton | Characterizing the metabolic perturbations induced by activity-based anorexia in the C57Bl/6 mouse using 1H NMR spectroscopy[END_REF] . We can speculate that several metabolites such as short-chain fatty acids, indole or ClpB could be involved [START_REF] Collins | The interplay between the intestinal microbiota and the brain[END_REF] . Indeed, it was recently reported that in response to a chronic stress, indole increased emotional responses [START_REF] Mir | The gut microbiota metabolite indole increases emotional responses and adrenal medulla activity in chronically stressed male mice[END_REF] and that the bacterial ClpB protein, a mimetic of αMSH neuropeptide, contributed to the response to the ABA model [START_REF] Dominique | Changes in Microbiota and Bacterial Protein Caseinolytic Peptidase B During Food Restriction in Mice: Relevance for the Onset and Perpetuation of Anorexia Nervosa[END_REF] . To better understand the central regulation of behavior in our conditions, we studied dopaminergic and serotoninergic systems in the hippocampus by evaluating mRNA levels of their receptors, as well as TNFα and BDNF. Again, we only observed a response to antibiotics treatment during ABA model in male mice. Indeed, for DRD1, DRD2, 5HT1R and TNFα, a statistical interaction between ABA and antibiotics (2-way ANOVA) was observed. ABA-induced increases of DRD1, DRD2 and TNFα were blunted by antibiotics. The role of these alterations in the behavioral changes during ABA model remains to be further studied. In female mice, antibiotics did not affect these parameters in response to ABA model. The role of sex hormones that were able to affect bacterial ClpB protein levels or other bacterial metoabolites in response to host starvation 54 should be deciphered.

In conclusion, we report here for the first time that antibiotics-induced gut microbiota depletion limits body weight loss and modifies body composition in response to the ABA model, in a similar manner in female and male mice. By contrast, gut microbiota depletion affects behavioral responses to the ABA model only in male mice. We anticipate that our results will pave the way for further studies aiming at better understanding the underlying mechanisms and at targeting gut microbiota in order to improving different facets of anorexia pathophysiology.

Acknowledgements

This

Figure 1 :

 1 Figure 1: Faecal bacteria depletion induced by antibiotics in male mice. Qquantification of bacterial density in mouse faeces at day 0 (d0) and day 15 (d15) in ATB-treated control (CT, open bars) and ATB-treated ABA (blue bars) male mice. Exact p-values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: ***, p<0.001 vs day 0.

Figure 2 :

 2 Figure 2: Body weight changes, body composition and anticipatory activity in male mice. Body weight changes from day 16 to day 28 (A.), in control (CT, closed circles), ATB-treated CT (open circles), and in ABA (closed triangles) and ATB-treated ABA (open triangles) male mice. Body weight changes at day 28 (B.), fat mass (C.), lean mass (D.) and anticipatory activity (E.). Column bar graphs show CT and ATB-treated CT male mice (open bars) and ABA and ATB-treated ABA male mice (blue bars). In each panel, the boxed text represents the results of the 2-way ANOVA (n=6-8/per group for body composition and n=13-16/per group for anticipatory activity). The p-value of the underlined and bold factor is lower than 0.05. Exact p-values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 vs CT or ATB-treated CT, # , p<0.05 vs untreated (no ATB) mice and $ , p<0.05 vs day 5.

Figure 3 :

 3 Figure 3: Plasma leptin, adiponectin and FITC-dextran concentrations in male mice. Plasma Leptin (A.), adiponectin (B.) and FITC-dextran 4kDa (C.) levels measured at day 28 in control (CT) and ATBtreated CT male mice (open bars) and in ABA and ATB-treated ABA male mice (blue bars). In each panel, the boxed text represents the results of the 2-way ANOVA (n=13-16/per group for leptin and adiponectin n=6-8/group for FITC-dextran). The p-value of the underlined and bold factor is lower than 0.05. Exact p values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 vs CT or ATB-treated CT mice.

Figure 4 :

 4 Figure 4: Hypothalamic pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNA expression in male mice. NPY (A.) and POMC (B.) mRNA expression at day 28 in control (CT) and ATB-treated CT (open bars) and in ABA and ATB-treated ABA (blue bars). In each panel, the boxed text represents the results of the 2-way ANOVA (n=13-16/per group). The p-value of the underlined and bold factor is lower than 0.05. Exact p-values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 CT vs ABA, # , p<0.05 ATB-treated CT vs ATB-treated ABA mice.

Figure 5 :

 5 Figure 5: Results of Open field test and quantification of hypothalamic cortico-releasing hormone (CRH) mRNA levels in male mice. Time (A.) travelled distance (B.), and travelled distance over time ratio (C.) at the center of the open field area measured at day 27 in control (CT) and ATB-treated CT (open bars) and in ABA and ATBtreated ABA (blue bars) male mice. Time (D.) travelled distance (E.) and travelled distance over time ratio (F.) at the periphery of the open field area measured at day 27. Immobility time (G.) and vertical activity (H.) measured during the open filed test at day 27. Quantification of hypothalamic CRH (I.) mRNA expression at day 28 in male mice. In each panel, the boxed text represents the results of the 2-way ANOVA (n=6-8/per group for open field test and n=13-16/per group for CRH quantification). The p-value of the underlined and bold factor is lower than 0.05. Exact p values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 vs CT or ATB-treated CT, # , p<0.05 vs untreated (no ATB) mice.

Figure 6 : 5 -HT1A) and 4 ( 5 -

 6545 Figure 6: Hippocampal dopaminergic receptor 1 (DRD1) and 2 (DRD2), serotoninergic receptor 1 (5-HT1A) and 4 (5-HT4), tumor necrosis factors α (TNFα) and brain-derived neurotrophic factor (BDNF) mRNA expression in male mice. DRD1 (A.) DRD2 (B.) 5-HT1A (C.) 5-HT4 (D.) TNFα (E.) and BDNF (F.) mRNA expression at day 28 in control (CT) and ATB-treated CT (open bars) and in ABA and ATB-treated ABA (blue bars) male mice. In each panel, the boxed text represents the results of the 2-way ANOVA (n=13-16/per group). The pvalue of the underlined and bold factor is lower than 0.05. Exact p-values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 CT vs ABA, # , p<0.05 ATB-treated CT vs ATB-treated ABA mice.

Figure 7 :

 7 Figure 7: Body weight changes, body composition and anticipatory activity in female mice. Body weight changes from day 16 to day 28 (A.), in control (CT, closed circles), ATB-treated CT (open circles), ABA (closed triangles) and in ATB-treated ABA (open triangles) female mice. Body weight changes (B.) at day 27, fat mass (C.), lean mass (D.) and anticipatory activity (E.). Column bar graphs show CT and ATB-treated CT female mice (open bars) and ABA and ATB-treated ABA female mice (red bars). In each panel, the boxed text represents the results of the 2-way ANOVA (n=6-8/per group). The p-value of the underlined and bold factor is lower than 0.05. Exact p-values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 vs CT or ATBtreated CT, # , p<0.05 vs untreated (no ATB) mice and $ , p<0.05 vs day 5.

Figure 8 :

 8 Figure 8: Plasma leptin, adiponectin and FITC-dextran concentrations in female mice.

Figure 9 :

 9 Figure 9: Results of Open field test and hypothalamic cortico-releasing hormone (CRH) mRNA expression in female mice. Time (A.) travelled distance (B.), and travelled distance over time ratio (C.) at the center of the open field area measured at day 27 in control (CT) and ATB-treated CT (open bars) and in ABA and ATBtreated ABA (blue bars) female mice. Time (D.) travelled distance (E.) and travelled distance over time ratio (F.) at the periphery of the open field area measured at day 27. Immobility time (G.) and vertical activity (H.) measured during the open filed test at day 27. Quantification of hypothalamic CRH (I.) mRNA expression at day 28 in female mice. In each panel, the boxed text represents the results of the 2-way ANOVA (n=6-8/per group for open field test and for CRH quantification). The pvalue of the underlined and bold factor is lower than 0.05. Exact p-values are displayed in Supplemental table 1. Results of Bonferroni posthoc tests are shown: *, p<0.05 vs CT or ATB-treated CT.

  Figure 1

  Figure 4

  Figure 8

Table 1 : Primer sequences for qPCR

 1 work was co-supported by the Microbiome foundation, the Roquette foundation for health and by European Union and Normandie Regional Council. Europe gets involved in Normandie with European Regional Development Fund (ERDF). PT received the support of the university of Rouen 34. Steinhausen, H.-C. & Jensen, C. M. Time trends in lifetime incidence rates of first-time diagnosed anorexia nervosa and bulimia nervosa across 16 years in a danish nationwide psychiatric registry study. Int. J. Eat. Disord. 48,845-850 (2015). Gut microbiota composition in male rat models under different nutritional status and physical activity and its association with serum leptin and ghrelin levels.

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