Mir-Farzin Mashreghi
email: mashreghi@drfz.de

B D'anna

Claudia Haftmann

Evridiki Sgouroudis

Anja Andrea Kühl

Ahmed Nabil Hegazy

Rene Riedel

Michael Flossdorf

Jun Dong

Franziska Fuhrmann

Anna-Barbara Stittrich

Isabel Panse

Gitta Anne Heinz

Zhuo Fang

Na Li

Ute Bissels

Farahnaz Hatam

Angelina Jahn

Ben Hammoud

Mareen Matz

Felix- Michael Schulze

Ria Baumgrass

Andreas Bosio

Hans-Joachim Mollenkopf

Joachim Grün

Andreas Thiel

Wei Chen

Thomas Höfer

Christoph Loddenkemper

Max Löhning

Hyun-Dong Chang

Nikolaus Rajewsky

Andreas Radbruch

MicroRNA-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes

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

Activation of the T cell receptor (TCR) by antigen and costimulation via CD28 induces proliferation and IL-2 production of CD4 + helper T lymphocytes [START_REF] Iezzi | The duration of antigenic stimulation determines the fate of naive and effector T cells[END_REF] . Once instructed to proliferate, the clonal expansion of helper T lymphocytes becomes independent of further antigenic stimulation [START_REF] Iezzi | The duration of antigenic stimulation determines the fate of naive and effector T cells[END_REF][START_REF] Van Stipdonk | Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation[END_REF] and is promoted by para-or autocrine IL-2 [START_REF] Jelley-Gibbs | Two distinct stages in the transition from naive CD4 T cells to effectors, early antigen-dependent and late cytokine-driven expansion and differentiation[END_REF] . To initiate and perpetuate helper T cell proliferation, T cell activation signals such as TCR-CD28 and IL-2 receptor (IL-2R) signaling have to inhibit Foxo1, which blocks cell cycle progression in resting cells via induction of p27 kip 4 . Foxo1 also induces the pro-apoptotic protein Bim [START_REF] Peng | Foxo in the immune system[END_REF] which is blocked by Bcl-2 in resting but not in activated helper T lymphocytes [START_REF] Pellegrini | Loss of Bim increases T cell production and function in interleukin 7 receptor-deficient mice[END_REF] . In the initial phase of activation Foxo1 protein is rapidly inactivated by post-translational modification, i.e. phosphorylation, dependent on TCR-CD28 signaling. This post-translational modification leads to exclusion of Foxo1 from the nucleus and promotes its functional inactivation [START_REF] Peng | Foxo in the immune system[END_REF][START_REF] Fabre | Stable activation of phosphatidylinositol 3-kinase in the T cell immunological synapse stimulates Akt signaling to FoxO1 nuclear exclusion and cell growth control[END_REF] . However, TCR and IL-2R signaling are short-lived and it has been unclear as to how Foxo1 is repressed in the late phase of clonal expansion.

Here we show that in the late phase of clonal expansion expression of Foxo1 is regulated post-transcriptionally by miR-182, which is a member of the miR-183-96-182 cluster [START_REF] Xu | MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster[END_REF] and is induced through IL-2R signaling. Activated signal transducer and activator of transcription 5 (STAT5) binds directly to the regulatory region of miR-182 and induces its expression. MiR-182 binds to a specific site of the 3'UTR of Foxo1 mRNA and reduces its expression. In activated helper T lymphocytes we found that specific inhibition of miR-182 results in increased amounts of Foxo1 and decreased clonal expansion. Constitutive overexpression of miR-182 in activated helper T lymphocytes results in drastically increased clonal expansion. In a transfer model of ovalbumin (OVA)-induced arthritis, inhibition of miR-182 in helper T lymphocytes reduced disease severity. Thus, our results indicate a critical contribution of miR-182 in the regulation of helper T cell expansion.

.

RESULTS

MiR-182 is induced in activated helper T cells

Murine naive (CD4 + CD62L hi CD25 -) helper T cells were activated in vitro under T helper (T H ) 1, T H 2 and T H 17 polarizing conditions. Expression of miR-96, miR-182 and miR-183 was determined by quantitative PCR. MiR-96 was not detectable, neither in naive nor in activated cells (data not shown). MiR-182 and miR-183 were both expressed in naive and in activated cells (Fig. 1a). In naive cells, expression level of miR-182 was threefold higher than those of miR-183. Six days after activation, expression of miR-182 and miR-183 was drastically upregulated in helper T lymphocytes of all culture conditions relative to naive helper T cells (Fig. 1a).

Expression of miR-182 was induced 50-to 200-fold and miR-183 expression increased 20-to 50-fold.

Irrespective of the polarizing culture conditions, expression of miR-182 increased between day one and two, reached a peak expression around day three (Fig. 1b) and decreased thereafter (Supplementary Fig. 1a). Non-activated naive CD4 + T cells did not upregulate miR-182 expression (Fig. 1b). There was no significant difference between miR-182 expression of T H 0, T H 1, T H 2 and T H 17 cells. Similar miR-182 kinetics were observed for human naive (CD45RA + ) and memory (CD45RO + ) helper T cells (Supplementary Fig. 1b,c). The strong induction of miR-182 and miR-183 suggests a critical role of these miRNAs in activated helper T cells.

Foxo1 is a target of miR-182 in activated helper T cells

Computational target prediction using PicTar 8 and TargetScan5.1 [START_REF] Lewis | Prediction of mammalian microRNA targets[END_REF] algorithms revealed Foxo1 as a putative target for the miRNAs of the miR-183-96-182 cluster.

To validate Foxo1 mRNA as a target of miR-96, miR-182 and/or miR-183, we cloned regions of the 3'UTR (>3000 nucleotides) of Foxo1 mRNA, containing (i) the predicted conserved overlapping binding sites for miR-96 and miR-182 and the binding site for miR-183 (nucleotides 1 to 174), (ii) only the miR-183 binding site (nucleotides 55 to 132) or (iii) only the overlapping miR-96 and 182 binding sites (nucleotides 105 to 191) into a retroviral vector downstream of a reporter gene encoding truncated human CD4 (Supplementary Fig. 2).

Murine naive CD4 + helper T cells were activated for 36 h and transduced with the retroviral vectors. Reporter gene expression was suppressed by 20-40% over a culture period of four weeks in cells carrying the vectors containing the predicted overlapping miR-96-miR-182 binding site, but not in cells carrying the vector containing only the predicted miR-183 binding site (Fig. 2a).

The functional relevance of the predicted overlapping miR-96-miR-182 binding site was validated by mutating its sequence from GUGCCAAA to GACGGUAA. The observed suppression of reporter gene expression could be reverted in this mutant (miR-96-miR-182 mut bs) (Fig. 2b). These experiments identify Foxo1 as a target of miR-182 in activated helper T lymphocytes. In these cells, Foxo1 is targeted only by miR-182 but not by miR-96 which is not expressed.

Dynamics of regulation of Foxo1 expression

We quantified Foxo1 mRNA and miR-182 in activated murine and human helper T cells. As expected, the levels of miR-182 and Foxo1 mRNA showed a negative correlation in murine (Fig. 3a) and in human helper T cells (Supplementary Fig. 3), with increasing miR-182 and decreasing Foxo1 mRNA over time.

To further confirm the interaction of miR-182 and Foxo1 we inhibited miR-182, by using either Locked Nucleic Acid (LNA) oligomers [START_REF] Orom | LNA-modified oligonucleotides mediate specific inhibition of microRNA function[END_REF] or antagomir oligonucleotides, both binding and inhibiting the mature miRNA. LNA-182 treatment of activated helper T cells blocked miR-182 expression by 60%, while Foxo1 mRNA increased by 20% and Foxo1 protein increased by 50% (Supplementary Fig. 4a-c). With antagomirs miR-182 could be blocked by more than 90% (Fig. 3b), while Foxo1 mRNA increased by 60% and Foxo1 protein increased by 50% (Fig. 3c,d).

Phosphorylated Foxo1 is exported from the nucleus and is therefore inactive [START_REF] Peng | Foxo in the immune system[END_REF] . To evaluate the kinetics of Foxo1 phosphorylation (P-Foxo1), we immunoblotted activated helper T cells. P-Foxo1 was not detectable before T cell activation (data not shown), but was rapidly induced by activation, already after 1 h. The ratio of total Foxo1 to P-Foxo1 remained low for 66 h (Fig. 3e). Thereafter, the ratio of total Foxo1 to P-Foxo1 increased about fourfold for the rest of the observation period, until 112 h after onset of activation (Fig. 3e). The amounts of total Foxo1 protein decreased gradually and remained low until 112 h after activation (Fig. 3f). These results indicate that suppression of Foxo1 in activated helper T lymphocytes is mediated initially by post-translational modification, until about 66 h after onset of activation, and thereafter by transcriptional and/or post-transcriptional regulation.

IL-2 is required for the induction of miR-182

Naive helper T cells were activated in vitro, in the presence or absence of recombinant IL-2. IL-2 induced expression of miR-182 in a dose-dependent fashion (Fig. 4a). Conversely, expression of miR-182 was inhibited by 80% by blocking IL-2 and CD25 (IL-2 receptor α chain) with antibodies (Fig. 4b), suggesting that IL-2R signaling promotes miR-182 expression.

IL-2R signaling activates STAT5 [START_REF] Rochman | New insights into the regulation of T cells by gamma(c) family cytokines[END_REF] . Interestingly, the intergenic miR-183-96-182 cluster contains several phylogenetically conserved binding sites for STAT5. Upon activation of naive helper T cells, STAT5 binding to a predicted STAT5 binding site could be determined by chromatin immunoprecipitation (ChIP) (Fig. 4c and Supplementary Fig. 5a-c). Specific inhibition of STAT5 activity with the chromonebased inhibitor nicotinyl hydrazone (compound 6) [START_REF] Muller | Discovery of chromone-based inhibitors of the transcription factor STAT5[END_REF] , prevented induction of miR-182 in activated helper T cells (Fig. 4d) without altering IL-2 production (Supplementary

Fig. 5d).

To analyze accessibility of the miR-182 locus, histone acetylation was determined by ChIP with antibodies specific for hyper-acetylated histone 4 (Ac-H4) under T H 1, T H 2 and T H 17 polarizing conditions. The miR-182 locus was hyper-acetylated after 24 h in activated naive helper T lymphocytes, pointing to a transcriptionally accessible conformation of the locus at this time. Subsequently, the hyper-acetylation declined gradually until it reached levels observed in naive cells after six days (Fig. 4e).

We wondered whether IL-2 signaling was sufficient to induce miR-182. Naturallyoccurring regulatory helper T cells (nT Reg ) do not produce IL-2 upon activation, but express the IL-2R constitutively [START_REF] Paust | Regulatory T cells and autoimmune disease[END_REF] . In these cells, expression of miR-182 could be induced by stimulation with recombinant IL-2, anti-CD3 and anti-CD28, but not by stimulation with recombinant IL-2 alone (Supplementary Fig. 5e). Thus, induction of miR-182 expression in helper T cells requires both TCR-CD28 and IL-2R signaling.

MiR-182 promotes clonal expansion of helper T cells

The role of miR-182 in helper T cell expansion was analyzed in loss and gain of function experiments. Treatment of activated helper T lymphocytes in vitro with inhibitory antagomir-182 reduced the number of viable cells by 50% on day three and by 60% on day four, compared to a scrambled control (antagomir-scr) (Fig. 5a).

The observed difference in cell numbers was due to increased cell death (Fig. 5b) and reduced proliferative capacity (Fig. 5c).

To test whether the treatment with antagomir-182 reduced helper T cell expansion in vivo, we treated ovalbumin (OVA)-specific helper T lymphocytes from DO11.10 mice with antagomirs and labeled them with carboxyfluorescein diacetate succinimidyl ester (CFSE) ex vivo. Subsequently, the helper T cells were transferred into Balb/c mice. One day after transfer, mice were immunized with OVA. On day four after immunization, lymph nodes and spleens of the mice were harvested and helper T cells analyzed. The number of viable helper T lymphocytes was reduced by 70% after antagomir-182 treatment (Fig. 5d). The CFSE labeling revealed a clear reduction in proliferation after antagomir-182 treatment (Fig. 5e).

Inhibition of miR-182 with LNAs led to a reduction of viable cells by 30%

(Supplementary Fig. 4d). A similar effect could be achieved by blocking the miR-treatment reduced the number of viable helper T cells by 20% (Supplementary Fig. 4d).

The effect of constitutive overexpression of miR-182 was tested in vivo with TCRtransgenic helper T lymphocytes from SMARTA mice that are specific for a lymphocytic choriomeningitis virus (LCMV)-derived peptide [START_REF] Oxenius | Functional in vivo MHC class II loading by endogenously synthesized glycoprotein during viral infection[END_REF] . Activated SMARTA Thy1.1 + T cells were transduced with a retroviral vector encoding miR-182 (miR-182RV), a scrambled control vector (scrRV), or an empty vector (RV) (Supplementary Fig. 6a). Expression of miR-182 was 40-fold higher in miR-182RVthan in scrRV-transduced helper T cells (Supplementary Fig. 6b). Transduced SMARTA Thy1.1 + helper T cells were then adoptively transferred into naive C57BL/6 mice. On day six after transfer mice were infected with LCMV. On day eleven after transfer the expansion of Thy1.1 + helper T cells was analyzed. MiR-182RVexpressing helper T cells exhibited a six-to fourteen-fold increased expansion relative to scrRV or empty RV transduced helper T cells, respectively (Supplementary Fig. 6c).

MiR-182 inhibition reduces OVA-induced arthritis in mice

As inhibition of miR-182 reduced clonal expansion of helper T cells in vitro and in vivo, we next analyzed whether miR-182 inhibition impacts on the ability of helper T cells to induce inflammation in a murine model of arthritis. OVA-specific helper T lymphocytes from OT-II mice were treated with antagomir-182 ex vivo and adoptively transferred into Rag -/-mice. One day after transfer, mice were immunized with OVA.

On day seven after transfer, OVA was injected into the left knee joint and knee swelling determined. Knee swelling was reduced by about 35% in the mice that received antagomir-182-treated helper T cells compared to control mice that received untreated cells (Fig. 6a). On day 15 after OVA challenge, the absolute numbers of CD4 + lymphocytes were determined in popliteal and inguinal lymph nodes. Those mice which received antagomir-182-treated cells had 60% fewer viable CD4 + lymphocytes (Fig. 6b).

The same experimental set-up was used to induce arthritis in Balb/c mice which were transferred with antagomir-182-treated helper T cells from DO11.10 mice. Knee swelling was reduced by about 40% with antagomir-182 treated helper T cells (Fig.

6c).

MiR-182 knockdown with antagomirs resulted in significantly lower histological scores of inflammation and tissue destruction (Fig. 6d, e and Supplementary Table 1). Infiltration of granulocytes and mononuclear cells into the inflamed tissue of the knee joint, bone and cartilage destruction and the formation of pannus were diminished in the mice with antagomir-182-treated cells compared to the control group (Fig. 6e).

DISCUSSION

In helper T lymphocytes the switch from the resting state in the absence of antigen, to proliferative expansion upon antigenic activation is tightly controlled by the transcription factor Foxo1 [START_REF] Stahl | The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2[END_REF] . Resting helper T cells express Foxo1, which induces interleukin-7 receptor (IL-7R), Bim and p27 kip [START_REF] Stahl | The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2[END_REF][START_REF] Dijkers | Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1)[END_REF][START_REF] Dijkers | Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1[END_REF][START_REF] Brunet | Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor[END_REF][START_REF] Ouyang | An essential role of the Forkhead-box transcription factor Foxo1 in control of T cell homeostasis and tolerance[END_REF][START_REF] Kerdiles | Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor[END_REF] and, in a positive feedback loop, the expression of itself [START_REF] Essaghir | The transcription of FOXO genes is stimulated by FOXO3 and repressed by growth factors[END_REF][START_REF] Al-Mubarak | Synaptic NMDAR activity suppresses FOXO1 expression via a cis-acting FOXO binding site: FOXO1 is a FOXO target gene[END_REF] . IL-7R signaling induces Bcl-2 to neutralize the proapoptotic effects of Bim 5 , whereas p27 kip blocks proliferation [START_REF] Rowell | Opposing roles for the cyclin-dependent kinase inhibitor p27kip1 in the control of CD4+ T cell proliferation and effector function[END_REF][START_REF] Wells | T cell effector function and anergy avoidance are quantitatively linked to cell division[END_REF] . Thus, to enable proliferation following antigenic stimulation, Foxo1 is blocked in activated helper T cells [START_REF] Stahl | The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2[END_REF] . IL-2 plays an important, yet not fully understood role in regulating clonal expansion of activated helper T cells [START_REF] Rochman | New insights into the regulation of T cells by gamma(c) family cytokines[END_REF] .

Immediately upon T cell activation, Foxo1 is blocked by TCR-dependent posttranslational phosphorylation [START_REF] Peng | Foxo in the immune system[END_REF] . However, TCR signaling ceases quickly and is decreased by 90% after 8 h of activation, while helper T cells continue to proliferate [START_REF] Naramura | c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligandinduced TCR down-modulation[END_REF][START_REF] Valitutti | Serial triggering of many Tcell receptors by a few peptide-MHC complexes[END_REF][START_REF] Salio | Agonist-induced T cell receptor down-regulation: molecular requirements and dissociation from T cell activation[END_REF] . In this phase of clonal expansion, proliferation and survival of the cells are dependent on IL-2 [START_REF] Jelley-Gibbs | Two distinct stages in the transition from naive CD4 T cells to effectors, early antigen-dependent and late cytokine-driven expansion and differentiation[END_REF] The 60-70% relative contribution of the IL-2-miR-182 axis to clonal expansion of activated helper T cells reflects the final phase of expansion when neither TCR nor IL-2R signaling are available. In the preceding phase, IL-2R signaling induces miR-182 but it also directly regulates Foxo1 expression by inducing phosphorylation of Foxo1 via phosphatidylinositol 3-kinase (PI3K) and Akt [START_REF] Peng | Foxo in the immune system[END_REF] . In the initial phase, TCR-CD28 signaling induces IL-2R and also regulates Foxo1 expression directly, via the PI3K-Akt pathway [START_REF] Peng | Foxo in the immune system[END_REF] . During ex vivo activation of naive helper T cells, the TCR-CD28dependent phase lasts for about one day, the directly IL-2-dependent phase lasts from day one to day three, while, as we show here, the IL-2-miR-182-dependent phase lasts from day two to day four. Both, the post-translational regulation by TCR-CD28 and IL-2, and the post-transcriptional regulation by miR-182 result in a disruption of the positive feedback loop of Foxo1 expression [START_REF] Essaghir | The transcription of FOXO genes is stimulated by FOXO3 and repressed by growth factors[END_REF][START_REF] Al-Mubarak | Synaptic NMDAR activity suppresses FOXO1 expression via a cis-acting FOXO binding site: FOXO1 is a FOXO target gene[END_REF] during clonal expansion. The regulation of miR-182 by IL-2 is obviously critical since helper T cells, after IL-2-driven clonal expansion, become IL-7-dependent for survival [START_REF] Dooms | Interleukin-2 enhances CD4+ T cell memory by promoting the generation of IL-7R alpha-expressing cells[END_REF] and expression of the IL-7R is dependent on Foxo1 [START_REF] Ouyang | An essential role of the Forkhead-box transcription factor Foxo1 in control of T cell homeostasis and tolerance[END_REF][START_REF] Kerdiles | Foxo1 links homing and survival of naive T cells by regulating L-selectin, CCR7 and interleukin 7 receptor[END_REF] . It is suggestive that prolonged downregulation of Foxo1 by constitutive expression of miR-182 leads to extinction of clonally expanded helper T cells.

In view of the critical role of STAT5 signaling for miR-182 induction, it remains enigmatic why only IL-2R signaling, but not IL-7R signaling induces miR-182. Both cytokines belong to the common cytokine receptor γ chain family and can activate STAT5 [START_REF] Rochman | New insights into the regulation of T cells by gamma(c) family cytokines[END_REF] . As we show here, STAT5 is the transcription factor that binds to the miR-182 locus. However, our results also suggest that IL-2R signaling on its own cannot induce miR-182. This was determined for regulatory T cells but is apparently also true for the other T H subsets since TCR signaling is always required to change the accessibility of the miR-182 locus for STAT5. No putative binding sites for NFAT or NF-κB are present in the miR-183-96-182 cluster. Thus, the molecular correlate of this "accessibility" remains yet elusive.

MiR-182 is encoded in a cluster with miR-183 and miR-96 [START_REF] Xu | MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster[END_REF] . Interestingly, the Foxo1 3'UTR has overlapping binding sites for miR-96 and miR-182 and a binding site for miR-183 [START_REF] Xu | MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster[END_REF] . For regulation of Foxo1 protein expression in activated helper T cells, however, miR-96 does not play a role, since it is not expressed at detectable levels, neither in naive nor in activated cells. MiR-183 is expressed in naive and in activated helper T cells. However, the predicted miR-183 binding site within the Foxo1 3'UTR did not confer suppression to a reporter gene in activated helper T cells. This observation could be explained by the fact that putative target sites are announced according to their sequence but might be inaccessible in vivo due to the secondary and tertiary structure of the mRNA [START_REF] Long | Potent effect of target structure on microRNA function[END_REF] . A role for miR-182, but not miR-183 in posttranscriptional regulation of Foxo1 expression has also been shown for breast cancer cells. In these cells constitutive expression of miR-182 induces proliferation [START_REF] Guttilla | Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells[END_REF] .

It is known that miRNAs generally have multiple targets and miR-182 is also likely to target other mRNAs in addition to Foxo1 mRNA. The proliferative effects of miR-182 found in this study, however, are due to Foxo1-targeting since masking of the specific miR-182 binding site within the Foxo1 3'UTR reproduced the effects observed with the inhibition of miR-182.

MiR-182, as we show here, provides distinct and decisive regulation of IL-2-induced clonal expansion of activated helper T cells and their survival as expanded cells by means of post-transcriptional regulation of Foxo1 expression. Our results showing reduced severity of disease in a transfer model of OVA-induced arthritis open up a new therapeutic avenue for the control of unwanted helper T cell expansion in immune-mediated diseases by antagomirs [START_REF] Krutzfeldt | Silencing of microRNAs in vivo with 'antagomirs[END_REF] . Conversely, mimics of miR-182 could be useful to inhibit Foxo1 in expanded helper T cells and thus eliminate these cells [START_REF] Wang | MicroRNA-based therapeutics for cancer[END_REF] .

AUTHOR CONTRIBUTIONS

METHODS

Mice

C57BL/6, DO11.10, Balb/c, SMARTAxThy1.1, OT-II and Rag -/-mice were bred under pathogen-free conditions, handled in accordance with good animal practice as defined by the German animal welfare bodies and sacrificed by cervical dislocation.

All animal experiments were performed in accordance with institutional, state, and federal guidelines (Landesamt Für Gesundheit und Soziales, Berlin, Germany).

Cell culture

Murine naive (CD4 + CD62L hi CD25 -) T cells from spleens of 6-10 weeks old mice were purified and cultured as described previously [START_REF] Richter | Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell cycle progression[END_REF] . Unless stated otherwise, C57BL/6 mice were used. Naive helper T cells (6 × 10 5 cells/ml) were stimulated with platebound anti-CD3e (5 μg/ml, clone 145-2C11, Miltenyi Biotec), soluble anti-CD28 (4 μg/ml, clone 37.51, Miltenyi Biotec) and, if cells were cultivated > six days, CD90depleted and irradiated (30 Gy) APCs (CD4 -fraction). Th1, Th2 and Th17 lineages were induced as previously described [START_REF] Chang | Expression of IL-10 in Th memory lymphocytes is conditional on IL-12 or IL-4, unless the IL-10 gene is imprinted by GATA-3[END_REF][START_REF] Lexberg | Th memory for interleukin-17 expression is stable in vivo[END_REF] . Th0 cells were induced by adding anti-IFNγ (10 µg/ml, clone AN18.17.24), anti-IL-4 (10 µg/ml, clone 11B11) and anti-IL-12 (10 µg/ml, clone C17.8). Recombinant IL-2 (eBioscience), anti-IL-2Rα (anti-CD25, 50 µg/ml, clone pC61.5), anti-IL-2 (50 µg/ml, clone JES6-1A12) or STAT5 inhibitor (compound 6, 200 µM) [START_REF] Muller | Discovery of chromone-based inhibitors of the transcription factor STAT5[END_REF] ( were added as indicated.

Ex vivo human helper T lymphocytes were obtained from buffy coats from anonymous healthy adult donors, in accordance with local ethical committee approval, and stimulated as stated elsewhere [START_REF] Dong | IL-10 is excluded from the functional cytokine memory of human CD4+ memory T lymphocytes[END_REF] .

Quantitative PCR

Total RNA was isolated using Trizol reagent (Invitrogen). For miRNA qRT-PCR cDNA was prepared from 200 ng of total RNA with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). Real time PCR was carried out using TaqMan MicroRNA Assays (Applied Biosystems) according to the manufacturer`s recommendations. TaqMan U6 snRNA Assay (Applied Biosystems) was used to normalize expression values.

For mRNA qRT-PCR, 400 ng of total RNA were reverse transcribed using the Reverse Transcription Kit (Applied Biosystems). The following custom primers were used for SYBR Green based real-time PCR (Roche): murine Foxo1 fw, 5

Reporter assay

To generate reporter vectors, sequences from Foxo1-3'UTR were cloned downstream of the human cd4 gene into the pMSCV vector (BD Biosciences).

Retroviral supernatants were generated and T cells were transduced as described before [START_REF] Niesner | Autoregulation of Th1-mediated inflammation by twist1[END_REF] . Human CD4 expression was quantified by flow cytometry using fluorochrome-coupled anti-human CD4 antibody (clone TT1).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation was performed as described previously [START_REF] Chang | Expression of IL-10 in Th memory lymphocytes is conditional on IL-12 or IL-4, unless the IL-10 gene is imprinted by GATA-3[END_REF] , using either polyclonal anti-acetyl-HistoneH4 (5 µg/ml, Millipore) or polyclonal anti-STAT5A/STAT5B antibody mixture (5 µg/ml, R&D Systems). The amount of

Overexpression of miR-182

To generate the miR-182 overexpression vector miR-182RV a 347 bp fragment harboring pre-miR-182 was amplified using the following primers: pri-182 fw, 5'-

Inhibition of miR-182 activity

To inhibit miRNA-182 either naive helper T cells or helper T cells that were activated for 24 h, were treated with specific antagomir-like oligonucleotides (Dharmacon). T cells were resuspended in serum-free ACCELL medium (Dharmacon) with 1 µM antagomir-182 or antagomir-scr,.After 1 h of incubation at 37°C in 5% CO 2 RPMI medium supplemented with 1 µM antagomir was added in threefold excess and cells were cultivated as described above. As control, T cells were incubated for 1h in ACCELL medium without addition of antagomirs.

Alternatively, miR-182 activity was inhibited with specific LNAs (Exiqon) that either targeted miR-182 or masked the miR-182 binding site. Activated helper T cells were transfected by nucleofection (Lonza) on day two post activation according to manufacturer's recommendations. The final concentration of inhibitors was 1 µM.

To monitor proliferation, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) directly after sorting (according to [START_REF] Quah | Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester[END_REF] ).

Immunoblot

Immunoblotting was performed as described previously [START_REF] Mashreghi | Inhibition of dendritic cell maturation and function is independent of heme oxygenase 1 but requires the activation of STAT3[END_REF] . Membranes were probed with primary antibodies against total Foxo1 (clone C29-H4) and tyrosinphosphorylated Foxo1 (polyclonal, both from Cell Signaling) and β-actin (Santa Cruz) for normalization. Immunoreactive bands were detected by chemiluminescence using ECL reagent (GE Healthcare) and analyzed by densitometry with Fuji LAS 3000 software.

LCMV model

Naive helper T cells from SMARTAxThy1.1 mice were activated for 36 h and transduced with miR-182RV. GFP + CD4 + and GFP -CD4 + cells were sorted by FACS on day four post activation. A total amount of 5x10 4 cells was transferred i.v. into C57BL/6 mice. Six days after transfer mice were infected with 200 plaque forming units (pfu) LCMV-WE. On day five after infection mice were bled and analyzed for Thy1.1 + GFP + helper T cells.

OVA-induced arthritis model 2x10 [START_REF] Fabre | Stable activation of phosphatidylinositol 3-kinase in the T cell immunological synapse stimulates Akt signaling to FoxO1 nuclear exclusion and cell growth control[END_REF] OVA-specific naive helper T cells from OT-II mice and DO11.10 mice were injected i.v. into RAG -/-recipients and Balb/c recipients, respectively. One day later recipients were immunized with cationized OVA (100 µg in 100 µl PBS/ mouse) at the tail base. The RAG -/-recipients were challenged on day seven after transfer, while the Balb/c recipients were challenged on day fourteen after transfer. For rechallenge cationized OVA (60 µg) was injected i.a. in the left knee joint. Knee swelling was determined as delta swelling between the left knee joint and the contralateral control knee joint. Knee joints were processed as described before [START_REF] Niesner | Autoregulation of Th1-mediated inflammation by twist1[END_REF] .

Statistics

All statistical analyses used the Student's t-test for paired or unpaired data, unless stated otherwise.

  . An understanding of Foxo1 regulation in the IL-2dependent phase of expansion has been elusive. Here we show that IL-2-induced miR-182 regulates Foxo1 at the post-transcriptional level, uncovering a further mechanism by which IL-2 can function as a T cell growth factor. Ectopic overexpression of miR-182 in helper T lymphocytes resulted in an about tenfold enhanced clonal expansion, demonstrating the potential of proliferative regulation at the post-transcriptional level| Ectopic expression, however, might overestimate the relative contribution of miR-182 to the regulation of clonal expansion. Without physiological downregulation of miR-182, inhibition of Foxo1 leads to a situation where all the expanded cells die (data not shown), probably due to a lack of common γ-chain signals provided by IL-2 or IL-7. Blocking physiological expression of miR-182 with antagomirs reduced clonal expansion by 60% in vitro and by 70% in vivo. This is in accordance with data from Dicer-deficient helper T cells which showed a 75% reduction in clonal expansion 28 . Thus, the IL-2-miR-182 axis constitutes an essential contribution to clonal expansion of activated helper T cells. Predictably, this contribution depends on the dynamics of IL-2 signaling during expansion. It remains to be determined to what extent this axis contributes to clonal expansion of memory cells, some of which express little if any IL-2 on their own, e.g. T H 2 cells and T H 1 cells 29 .

A

  .B.S. designed and performed experiments, analyzed data and wrote the manuscript; C.H. did experiments and analyzed data; E.S. designed and performed the OVA-induced arthritis experiments; A.K. and C.L. performed histology; A.N.H., I.P. and M.L. designed and analyzed LCMV experiments; R.R. performed the OVAinduced arthritis experiments; M.F. and T.H. analyzed CFSE measurements; J.D. and A.T. did human T cell experiments; F.F. provided cells; G.A.H. did inhibitory experiments; N.L., Z.F., U.B., A.B., J.G., W.C. and N.R. did miRNA screening experiments and/or analyzed them; N.R., M.L. and Z.F. discussed the results and commented on the manuscript; F.H. revised the manuscript; A.J. constructed scrambled overexpression vector; B.H., M.M., F.M.S. and R.B. provided technical support and conceptual advice; H.D.C. designed experiments and supervised research; A.R. designed study, supervised research and wrote the manuscript; M.F.M. designed the study, analyzed data and wrote the manuscript.

Figure 1

 1 Figure 1 MiR-182 is induced upon activation of primary naive helper T

Figure 2

 2 Figure 2 Foxo1 is a target of miR-182. (a) Reporter assays using a reporter vector

Figure 3

 3 Figure 3 Inactivation of Foxo1 by phosphorylation occurs rapidly upon

Figure 4 IL- 2

 42 Figure 4 IL-2 induces miR-182 expression. (a) Expression of miR-182 in T H 0 lymphocytes that were activated with plate-bound anti-CD3 and anti-CD28 for 48 h

Figure 5

 5 Figure 5 Inhibition of miR-182 reduces T cell expansion in vitro and in vivo. (a)

Figure 6

 6 Figure 6 Inhibition of miR-182 reduces severity of OVA-induced arthritis in mice. (a,c) Knee swelling of OVA-immunized (a) Rag -/-mice that were adoptively

  immunoprecipitated DNA was determined by quantitative PCR. Putative binding sites in conserved regions of the miR-183/96/182 cluster (chr6:30115650-30119860 [-])were identified using the rvista webpage (http://rvista.dcode.org/), based on TRANSFAC professional V10.2 library. The following primers were used: pre-miR-182 fw, 5'-ACCGGGTCCCATTACCTTAC-3' and pre-miR-182 rev, 5'-GCCTCCTAAAACCACCCTA-3'; STAT5 fw, 5'-AGGGTGGCTAGAAGCCCTAA-3'

GTTAACGTTAACTGTGGGAAGAGCGC- 3 '

 3 (including 2 HpaI restriction sites for cloning) and pri-182 rev, 5'-CTCGAGAAAAAACACCGAGAAGAGGTCGA-3'. The pri-miR-182 fragment was cloned into the pQCXIX-vector (Clontech) under control of the U6 promoter. GFP was inserted under the control of the EF1α promoter, both derived from pLVTHM from D. Trono (École Polytechnique Fédérale de Lausanne).The control vector scrRV contains a scrambled version of miR-182 that was inserted of viral supernatants and T H cell transduction were performed as described for the reporter assays.

ACKNOWLEDGEMENTS

We thank Thorsten Berg (Max Planck Institute for Biochemistry, Martinsried, Germany) for providing the STAT5 inhibitor. A.B.S. and A.N.H. are supported by the GRK1121 of the German research council (DFG), C.H. is supported by the International Max Planck Research School for Infectious Diseases and Immunology. M.L. is a Lichtenberg fellow of the Volkswagen Foundation. This work was supported by the FORSYS program of the Federal ministry of education and research,, the SFB 618, SFB 650, SFB 633 and SFB TR52 of the DFG, and the EU MUGEN Network of

Excellence. We wish to thank Heidi Schliemann, Tuula Geske, Heidi Hecker-Kia, Anette Peddinghaus, Toralf Kaiser and Jenny Kirsch for excellent technical assistance. We thank Miltenyi Biotec for the miRXplore microarray experiments.


Guillaume Péron
email: guillaume.peron@cefe.cnrs.fr

Jean-Dominique Lebreton

Pierre-André Crochet

Péron

Costs and benefits of colony size vary during the breeding cycle in Black-headed Gulls Chroicocephalus ridibundus

Keywords: Breeding success, Chroicocephalus ridibundus, Coloniality, Density-dependence, Fecundity, Larus

We studied the effects of colony size on individual reproductive success in a multisite population of Black-headed Gulls where colony size ranged from 10 to 5000 pairs. By focusing on family size, the number of chicks attended by individually marked parents, and accounting for between-individual variation, we detected a negative colony-size effect during the very first days of life of the chicks that was compensated by a subsequent increase in the proportion of surviving chicks with colony size. We suggest that this result originates in the interplay between overcrowding costs acting on hatching success, and benefits of colonial breeding, most probably more efficient food-searching (foraging enhancement), acting on chicks" survival. However, the frequency of complete colony failure increased with decreasing colony size. Taking this hazard risk into account yielded a corrected estimate of the effect of colony size on breeding success, and indicated that the largest colonies were the most productive. This pattern is congruent with the previous finding that larger colonies are more attractive to dispersing breeders.

Introduction

Colonial breeding (breeding in high-density sites exempt of feeding resources with territories limited to the nest area) is quite widespread in birds and especially in seabirds. One of the striking patterns is the sheer variation that exists in colony size. A recurrent question among ornithologists is whether this variation simply reflects variation in ecological conditions (distribution of resources, availability of nest sites, etc.) or whether the choice to settle in a colony of a given size is a trait subject to selection. These questions are generally addressed by studying variation in reproductive success (RS), which appears to be a useful metric to study the fitness consequences and ecological correlates of colony size (but see [START_REF] Danchin | The evolution of coloniality: the emergence of new perspectives[END_REF].

In colonial birds, especially in Charadriiforms, provisioning rate is often considered among the main determinants of reproductive success [START_REF] Uttley | The effects of food abundance on breeding performance and adult time budgets of Guillemots Uria aalge[END_REF][START_REF] Jodice | Assessing the nutritional stress hypothesis: relative influence of diet quantity and quality on seabird productivity[END_REF]; see also [START_REF] Graves | Why does the Herring Gull lay 3 eggs[END_REF][START_REF] Hamer | The influence of foodsupply on the breeding ecology of Kittiwakes Rissa tridactyla In Shetland[END_REF][START_REF] Sadoul | Changes in the numbers and the distribution of colonial Charadriiformes breeding in the camargue, southern France[END_REF][START_REF] Van Dijk | Reproductie van de Kokmeeuw Larus ridibundus in kust-en binnenlandkolonies[END_REF]).

Provisioning rate depends on foraging success, which is known to be enhanced via information exchange between conspecifics foraging in the same area [START_REF] Ward | Importance of certain assemblages of birds as information-centers for food-finding[END_REF][START_REF] Clark | Foraging and flocking strategies -Information in an uncertain environment[END_REF][START_REF] Richner | Communal life: honest signaling and the recruitment center hypothesis[END_REF][START_REF] Brown | Coloniality in the Cliff Swallow[END_REF] see further detail in the discussion section). Provisioning rate is therefore expected to increase with conspecific density on the foraging grounds [START_REF] Pöysa | Group foraging in patchy environments -the importance of coarse level local enhancement[END_REF], and, as the density of foraging individuals increases around large colonies [START_REF] Brandl | How to live in colonies -Foraging range and patterns of density around a colony of Black-headed Gulls Larus ridibundus in relation to the gulls energy budget[END_REF], we expect an enhanced reproductive success in large colonies. However, some studies indicate the opposite effect, i.e., that competition forces individuals from large colonies to travel further and longer to find food than individuals from small colonies [START_REF] Lewis | Using behavioural and state variables to identify proximate causes of population change in a seabird[END_REF], sometimes to the point where colony size negatively impacts on body condition of both adults and chicks [START_REF] Santema | Does foraging efficiency vary with colony size in the fairy martin Petrochelidon ariel?[END_REF]. Furthermore, competition is only one of the known or supposed costs of breeding in large colonies. These costs also include higher rate of transmission of disease and parasites, higher levels of aggression and antagonistic behaviours, etc. [START_REF] Brown | Coloniality in the Cliff Swallow[END_REF][START_REF] Danchin | The evolution of coloniality: the emergence of new perspectives[END_REF].

It is thus not surprising that, among the large number of studies reviewed by Brown and Bomberger Brown (1996, p321-323), every pattern of variation in RS with colony size can be found, from no effect to a directional trend for a benefit to the largest or smallest colonies through a non linear effect with an optimal colony size. This variation among studies suggests that (i) the costs and benefits of colonial breeding are not the same in different systems and/or (ii) the observed patterns depend on the RS variable used and on the stage of the breeding season that is under study (proportion of failed nests, average clutch size, average number of fledglings per breeding pair, fledgling survival, etc.).

Our aim in this paper is to contribute to these debates by studying colony size effects on RS in a Black-headed Gull Chroicocephalus ridibundus (BHG) multisite population presenting a contrasted distribution of colony sizes. Indeed a single, large colony concentrated most pairs and increased in size during the study period (from around 2000 to around 5000 pairs), whereas several small colonies (numbering on average 163 pairs ±171) decreased in both number and total size. More precisely, we examined (i) how the costs and benefits of colony size interacted throughout chicks" development and (ii) whether a potential cost of breeding in small colonies could have driven the aggregation of breeders in the largest colony.

Although BHG are known to occasionally breed as solitary pairs there was no such record in the dataset. We therefore did not contrast solitary vs. colonial breeding in this study but we investigated the costs/benefits of colony size.

The data we used for that purpose was the family size, i.e. the number of chicks attended by individually-marked parents. By using its variation with chicks" age, we had access to two variables for RS:

-the initial family size (IFS), which was considered as a proxy for the hatching success and chicks" survival during the first few days, -the decrease in family size with age (DFS), which was considered as a proxy for chicks" survival rate during growth and until fledging. By using this variable instead of the family size just before fledging, we were able to use all the records of family size between hatching and fledging, hence to increase the sample size.

Predicting how IFS and DFS should vary with colony size in our BHG population required a few assumptions to be made on the main processes at stake. First, overcrowding costs in BHG are of three main kinds: increase in the level of competition for the access to high, secure nest locations, increase in alarm flight frequency and duration, and increase in antagonistic behaviours. Eggs and young chicks are supposedly more sensitive to these costs than older chicks: they can fall in the water or be predated from low, unsafe nests, they can die because of poor thermoregulation during alarm flights and they can suffer from aggression by neighbouring adults. In short, we assumed that overcrowding costs should impact more on IFS (eggs and young chicks) than on DFS (older chicks).

Second, the food resource available to chick-raising parents can vary with colony size, either positively through foraging enhancement, or negatively through density-dependence. If we make the assumption that food resource during the breeding season does not impact on clutch size and hatching success, we can predict that DFS but not IFS should be impacted by colony size effects on food resource (BHG as capital breeders, [START_REF] Drent | The prudent parent -Energetic adjustments in avian breeding[END_REF][START_REF] Hamer | The influence of foodsupply on the breeding ecology of Kittiwakes Rissa tridactyla In Shetland[END_REF] in another Larid species, but see [START_REF] Thyen | Effects of individual life-history traits and weather on reproductive output of Black-headed Gulls Larus ridibundus breeding in the Wadden Sea, 1991-97[END_REF] and further details in the discussion section). Overall from the two preceding points we thus predict that IFS should decrease with colony size and that DFS should increase with colony size.

Third, predation risk is expected to decrease with colony size, due to among others the dilution effect [START_REF] Hamilton | Geometry for the selfish herd[END_REF] and early-warning effect [START_REF] Lazarus | Early warning function of flocking in birdsexperimental study with captive Quelea[END_REF]). An increase with colony size in the efficiency of anti-predator behaviours (mobbing) also likely occurs [START_REF] Allainé | The effects of colony size and breeding stage on colony defence pattern in the Black-headed Gull[END_REF].Predators" distribution can, however, alter these predictions. (i) If predators are strongly localised (territorial, dependent on rare habitat features…), the associated risk can eventually be independent of colony sizes. (ii) Alternatively, predators can concentrate their attacks on the densest sites where preys may appear more available [START_REF] Larivière | Effect of density and nearest neighbours on simulated waterfowl nests: can predators recognize high-density nesting patches?[END_REF].

These simple predictions can be further complicated by environmental stochasticity due to climatic accidents [START_REF] Aebischer | Immediate and delayed effects of a gale in late spring on the breeding of the Shag Phalacrocorax aristotelis[END_REF], human perturbations [START_REF] Calado | Little Tern (Sterna albifrons) status and conservation at Ria Formosa Natural Park, Algarve, Portugal[END_REF] or predatordriven failures [START_REF] Ratcliffe | Modelling the benefits of American Mink Mustela vison management options for terns in west Scotland[END_REF]. For example, climates partially drives the RS of Fulmarus glacialis [START_REF] Lewis | Effects of extrinsic and intrinsic factors on breeding success in a long lived seabird[END_REF], which would probably have modified the effect of colony size in that system. In our BHG population, whole-colony failures, i.e. the desertion of a given colony before fledging, are relatively frequent (Table 1) and constitute a major external driver of RS. Their causes include man-driven droughts of the ponds and perturbations by terrestrial predators [START_REF] Lebreton | Fécondité de la mouette rieuse Larus ridibundus dans une colonie importante de la plaine du Forez (Loire, France)[END_REF]. We further suggest that once a threshold proportion of breeders have failed, the remaining pairs desert the colony as well. The threshold proportion is supposedly more easily reached in small colonies (e.g., a single wildboar Sus scrofa will destroy most nests in a 100-pair colony, but its impact will be less visible in a 4000-pair colony). Thereby the risk of whole-colony failure should decrease with colony size, providing an advantage for breeding in large colonies. We combined the results from the family size data introduced above with an estimate of the probability of whole-colony failure. This made it possible to correct the estimate of RS for the occurrence of such failures and see how they affect the relationship between colony size and RS during the early and late parts of the breeding cycle.

Methods

Study site and species

The study site is located in the Forez basin, Loire department, central France. Each year between 1978 and 2008, from 9 to 34 colonies (mean 19.9) of BHG bred in man-made ponds situated in a farmland mosaic (see [START_REF] Lebreton | Régulation par le recrutement chez la Mouette rieuse Larus ridibundus[END_REF] for a more complete description of the study area). Gulls lay a clutch with a modal size of three eggs from mid-April to the beginning of June [START_REF] Lebreton | Fécondité de la mouette rieuse Larus ridibundus dans une colonie importante de la plaine du Forez (Loire, France)[END_REF]. Chicks and adults have been ringed since 1978.

One of the colonies (La Ronze) differed from other colonies due to its size (between 2000 and 5000 pairs), while the other colonies numbered on average 163 pairs (SD: 171). Also, between 1978 and 2006 La Ronze was occupied continuously, contrary to smaller colonies which experienced either spontaneous desertion by the gulls and/or temporary man-induced perturbations that prevented breeding (droughts for management purpose). In 2007 La Ronze experienced such a drought; in 2008 the colony reappeared on that pond but numbered only 500 pairs.

Data collection -Family size data

Each year between 1979 and 2008 a subset of the active colonies (depending on access authorizations) was visited to look for ringed birds. We collected data simultaneously in La Ronze and in the smaller colonies but due to colony-specific laying dates more time was devoted to the large colony early in the season relative to small colonies. Field work lasted usually four or five weeks, between May 5 at the earliest and June 28 at the latest. However, data collection in the small colonies began only from 1995 onwards, with only a few sparse records before that year (see Table S1 in Appendix for details on when and where data were collected). We opportunistically recorded family size of individually-marked adult birds (metal rings on tibia) at various stages of the chicks" development while moving through the colonies in a floating blind [START_REF] Lebreton | Régulation par le recrutement chez la Mouette rieuse Larus ridibundus[END_REF]. The number of chicks per pair (= the family size) was counted when the parents were feeding the chicks. Chicks" age in days was estimated with an accuracy of 2 to 4 days through detailed observation of size and plumage development [START_REF] Lebreton | Fécondité de la mouette rieuse Larus ridibundus dans une colonie importante de la plaine du Forez (Loire, France)[END_REF]. As families were recorded while looking for ringed parents, it is unlikely that family size influenced on the detection probability of families. Of course, only families with at least one chick could be recorded (thereby excluding failed breeding attempts).

-Colonies" average RS Each year, we visited most of the colonies at least twice (installation period and end of breeding cycle), including those that were not surveyed for family sizes. We recorded the number of pairs (colony size) and the colonies" average RS, quantified using a three-level index: 0 (no fledgling produced), 1 (low success), 2 (high success). Whole-colony failure is hereafter the occurrence of a "0". This protocol provided us with an estimate of the proportion of birds that tried to breed even if they failed early and did not enter our dataset on family size.

Model for the family size data

The dataset included 1,255 family size records from 966 individually-marked birds over 31 years (Fig. 1). This dependent variable was worth 1, 2 or 3 (with a few marginal records of 4-chicks families possibly, but not compulsorily, due to chick adoption or intraspecific clutch parasitism). Generalized linear mixed models (GLMM) with Poissondistributed error terms and log link function were fitted using the restricted maximum likelihood (REML) method implemented in the macro GLIMMIX in SAS 9.1.

We included the fixed effects of chicks" age, colony size and their interaction.

Theoretically, the effect of colony size is not thought to be linear (see [START_REF] Clark | Foraging and flocking strategies -Information in an uncertain environment[END_REF][START_REF] Forbes | Habitat choice in breeding seabirds -When to cross the information barrier[END_REF], which was taken into account via a log-transformation of colony sizes. Similarly, chicks" death rate is expected to decrease with age, advocating the log-transformation of chicks" ages also. We corrected for the potential confounding effects of between-individual (966 individuals), -year (31 years) and -site (10 sites) variation with independent (i.e., non-nested) random effects. Temporal autocorrelation was accounted for using a first order autoregressive structure.

A large number of observers contributed to the data, but one of us (J.D.L.) contributed much more than other observers (collecting more than half of the data) and spent more time in the LR colony than other observers, but less time in the small colonies. Because old chicks are very mobile around their nest site, our method relied on a certain amount of subjectivity to decide when a family size had been accurately determined, suggesting non-negligible observer effect. We thus accounted for the observer effect with a fixed effect. Preliminary analyses indicated that contrasting J.D.L.to all other observers captured most of the betweenobserver variation.

We expected a low statistical power due to sparse data in several colony size × chicks" age cells. Statistically significant fixed effects were considered to be those whose confidence interval (CI) did not encompass zero. For the random effect structure we used the Akaike Infromation Criterion (AIC) to discard non-significant effects.

Model for the whole-colony failure data

We built a GLMM with binomial error term and logit link function, with occurrence of whole-colony failure as dependent variable and colony size as fixed effect, colony identity and year as random effects (with a temporal autocorrelation structure), using the restricted maximum likelihood (REML) method implemented in the macro GLIMMIX in SAS 9.1. This model produced an estimate of the probability of colony failure depending on colony size.

Statistical significance of the effects was determined as in the previous section.

Assuming independence between family size data and whole-colony failure data, and in order to obtain a model of RS accounting for colony size effects on both family size and the frequency of whole-colony failures, we multiplied the predictions from the two models constructed above. Confidence intervals on this expected RS were computed using a multiple (1000 times) resampling procedure, using the standard errors estimated at the previous stage to compute an expected distribution of models" parameters.

Results

Model for the family size data Fixed effects of colony size, chick"s age and their interaction all had confidence intervals only marginally encompassing zero, and were thus maintained in the final model (Table 2). Concerning random effects, year (with autoregressive structure) and individual effects proved significant with regards to AIC. The GLMM revealed an important variation between individuals: parent identity accounted for 20.4% of the total variance in the data.

Year effect was much lower (2.3% of the total variance). Table 2 gives details of the estimated parameter values in the final model. The statistically significant effect of colony size indicates that IFS (family size shortly after hatching) decreased with colony size (Fig. 2). This is partly compensated by a decrease in DFS (chick mortality) with colony size (interaction term in Table 2; Fig. 2). Observer effects are presented in Table S2 in Appendix; they were discarded due to statistical non-significance.

Model for the whole-colony failure data.

As expected from an examination of the raw data (Table 1) the probability of complete colony failure markedly decreased with increasing colony size (Table 3: fixed effect), even after controlling for the colony identity and year using random effects. Concerning random effects, the GLMM revealed a significant variation between years: year accounted for 32.8% of the total variance in the data. Random effect of colony identity was discarded by AIC. The temporal autocorrelation of colony failure probability appeared non-significant with regard to AIC and autoregression coefficient estimate.

Combining the two models.

The "composite" model of RS obtained by multiplying the predicted family size at fledging in successful families (from the model in Table 2) and the predicted probability of whole-colony failure (from the model in Table 3) suggested a strongly detrimental effect of breeding in colonies of 100-200 pairs or less but little effect of colony size on RS for colony size above 300 pairs (Fig. 3).

Discussion

Colony size effects on initial family size Our first hypothesis (colonial breeding is costly at the egg and young-chick stages) is verified by the fact that IFS decreased with colony size. Given that the most abundant predators (Carrion Crow Corvus corone, medium-sized raptors such as Black Kite Milvus migrans, herons, snakes, Canids, Mustelids) prey mostly on eggs or young chicks, explaining this result by predation risk reduction would imply that small colonies are less subject to predation, which seems improbable [START_REF] Hamilton | Geometry for the selfish herd[END_REF][START_REF] Lazarus | Early warning function of flocking in birdsexperimental study with captive Quelea[END_REF][START_REF] Allainé | The effects of colony size and breeding stage on colony defence pattern in the Black-headed Gull[END_REF], unless they attract less predators [START_REF] Larivière | Effect of density and nearest neighbours on simulated waterfowl nests: can predators recognize high-density nesting patches?[END_REF].

A second hypothesis is that females in large colonies have access to fewer resources and thus produce less and/or poorer-quality eggs than females in small colonies. Yet in the introduction we relied on the assumption that, at the egg-laying stage, females use mostly resources secured before settling in the colonies. This assumption that BHG are capital breeders [START_REF] Drent | The prudent parent -Energetic adjustments in avian breeding[END_REF] is however contradicted by [START_REF] Thyen | Effects of individual life-history traits and weather on reproductive output of Black-headed Gulls Larus ridibundus breeding in the Wadden Sea, 1991-97[END_REF] who found that in another BHG population clutch size changes with weather conditions during the breeding season.

A third explanation (that we consider the most likely) is that the larger the colony, the more intense the risk that eggs and young chicks are lost during alarm flights or during intense antagonistic behaviours. Here we thus assume that colony size and nest density are correlated (which seems relevant due to the much smaller variation in pond area compared to colony sizes). This is confirmed by our own observations (although no quantitative data are available yet) of more numerous fights among neighbours in large colonies and of occasional loss of eggs or small chicks during or after alarms because of such fights, or as small chicks that leave the nest during alarms are often attacked by neighbours when they cross their territories.

Even if we are unable to firmly choose among the potential explanations (resource limitation at the laying stage or increased loss of eggs and small chicks), our result that IFS decreases with colony size at least indicates that, at the egg-laying stage, overcrowding costs exceed any benefit of colony size.

Colony size effects on the decrease in family size with chicks" age Our second prediction, that DFS decreases with colony size, is supported by the interaction between colony size-and chicks" ageeffects. This interaction indicates that for each family the proportion of hatched chicks that survives to fledging increases with colony size. Any mechanism increasing chick survival but not hatching success can explain these results, and we consider here predation risk reduction and foraging enhancement.

Predation risk reduction mechanisms can include dilution effect [START_REF] Hamilton | Geometry for the selfish herd[END_REF] and early-warning effect [START_REF] Lazarus | Early warning function of flocking in birdsexperimental study with captive Quelea[END_REF]). An increase with colony size in the efficiency of antipredator behaviours (mobbing) also likely occurs [START_REF] Allainé | The effects of colony size and breeding stage on colony defence pattern in the Black-headed Gull[END_REF], thereby allowing individuals from large colonies to spend more time in other chick-care activities. However in the predation risk reduction scenario it seems hard to elucidate why the benefits act more on old chicks (DFS) than on eggs and very young chicks (IFS).

Foraging enhancement is therefore a more likely hypothesis explaining the observed increase in chicks" survival with colony size. Congruent with this consideration is the fact that the exchange of food information has been demonstrated in our study species [START_REF] Andersson | Food information in the Black-headed Gull, Larus ridibundus[END_REF] as well as in a closely related species that has similar feeding habits [START_REF] Evans | Foraging-flock recruitment at a Black-billed gull colony -implications for the information-center hypothesis[END_REF].

These two studies are to our knowledge the only two testing the existence of information transfers in Chroicocephalus gulls. Social foraging is also suggested by the finding in our study population that neighbours in the largest colony tend to forage together (Prévot-Julliard and Lebreton 1999), and by the consideration that white plumage makes it possible to detect successful foragers at a great distance [START_REF] Beauchamp | Social foraging and the evolution of white plumage[END_REF]. In the next paragraph we thus provide a few additional elements on foraging enhancement in the Forez BHG.

Locating food sources and foraging enhancement: a simplistic model If there was one and only one feeding site available at any time, and if they were foraging individually, gulls would have to patrol their home range of size S, at the speed V and with the detection distance R. The instantaneous probability λ that a lone bird without prior knowledge would find the feeding site is:

S RV 2
. With the plausible values S = 710km² (a circle of approx. 15km radius: Brandl and Gorke 1988), V = 11.3 m.s -1 (Shamoun-Baranes and van Loon 2006) and R = 500m, we obtain 0.057 h -1 , which would mean that a lone bird needs on average 17.5 hours to find an appropriate feeding site. The selection pressure to enhance foraging efficiency would thus be high. If N birds looked for the same site, and neglecting the time needed for information transfers, the instantaneous probability that the field is found would be N×λ [START_REF] Clark | Foraging and flocking strategies -Information in an uncertain environment[END_REF].

We must admit that the assumption of the uniqueness of the feeding site is quite unlikely. Nevertheless, this simplistic model highlights the underlying idea that finding the best food possible (i.e., securing the highest ratio energy or nutriments brought back to the chicks vs. time or energy spent by the parents) takes too much time if foraging solitarily.

Furthermore, information transfer can allow a rapid specialisation on the best available resource at any time. We suggest information transfers as a possible explanation for our result that chick survival increased with colony size.

Correcting for whole-colony failures

The family size at age 32 days corresponds to the number of fledglings per pair. Our results indicated that, excluding complete breeding failures, BHG in our population produced between 1.4 and 1.6 fledglings per pair depending on colony size (curve labelled "age 32" in Fig. 2). This value lies in the high part of the range of reported values for BHG (0.3 to 2 fledglings per pair [START_REF] Patterson | Timing and spacing of broods in the Black-headed Gull Larus ridibundus[END_REF][START_REF] Lebreton | Fécondité de la mouette rieuse Larus ridibundus dans une colonie importante de la plaine du Forez (Loire, France)[END_REF][START_REF] Viksne | Some problems in Black-headed Gull Larus ridibundus research and the necessity of international cooperation in this respect[END_REF][START_REF] Thyen | Effects of individual life-history traits and weather on reproductive output of Black-headed Gulls Larus ridibundus breeding in the Wadden Sea, 1991-97[END_REF][START_REF] Van Dijk | Reproductie van de Kokmeeuw Larus ridibundus in kust-en binnenlandkolonies[END_REF]). This result is probably due to our not accounting for reproductive failures (pairs that do not produce any fledgling on a given year).

Reproductive failures are partially accounted for when correcting the previous estimates for the occurrence of whole-colony failures. The much more frequent occurrence of whole-colony failures in small colonies (Table 1) is an additional cost of breeding in small colonies, and it appears as the main determinant of the sharp decrease in expected RS at low colony sizes (Fig. 3). This low RS in small colonies is congruent with previous results that such colonies are less often chosen by dispersing experienced breeders (as opposed to inexperienced breeders; [START_REF] Péron | Breeding dispersal in Black-headed Gull : the value of familiarity in a contrasted environment[END_REF], and that breeders from small colonies are less sitefaithful than breeders from large colonies (Péron et al. submitted). Whether these results stem from colony size per se or a correlation between size and "quality" remains debatable. The result that whole-colony failure probability is neither temporally autocorrelated nor dependent on colony identity suggests that whole-colony failures can be viewed as an external hazard, i.e., independent of the true quality of the colony site. However in a low-quality habitat individuals are expected to be less site-faithful which could be sufficient to maintain colonies at a small size, and therefore colony size and colony quality could match exactly.

Conclusion

The 32-year long BHG dataset that we studied illustrated that the various costs and benefits of colony size varied during the breeding cycle, with costs exceeding benefits at the beginning of the cycle and benefits exceeding costs at the end of the cycle. Moreover the perturbation regime (environmental stochasticity) appeared as a main driver of breeding success and largely modified the final output of breeding in the various colony sizes, with the smallest colonies being the most exposed to perturbations. These findings put an interesting light on the inconsistencies between previous studies (reviewed in [START_REF] Brown | Coloniality in the Cliff Swallow[END_REF]; see also [START_REF] Lewis | Effects of extrinsic and intrinsic factors on breeding success in a long lived seabird[END_REF][START_REF] Santema | Does foraging efficiency vary with colony size in the fairy martin Petrochelidon ariel?[END_REF]. They suggest that unaccounted differences in the regime of perturbation might have flawed previous results. Any interaction between colony size and the regime of perturbation can be predicted, with increases in the rate of perturbation with colony size possibly stemming from predators" attraction towards aggregated prey items, and decreases possibly originating from a threshold mechanism, as is suggested in our system, or from small colonies being settled in lower-quality habitats [START_REF] Forbes | Habitat choice in breeding seabirds -When to cross the information barrier[END_REF]. Eventually the estimates of RS along the colony-size gradient in our system provided an explanation for the observed dynamics of our population. It seems indeed likely that individuals aggregated in the largest colony because this colony provided the best RS.

Density-dependent mechanisms did not counter-balance this benefit. Results concerning breeding dispersal are in line with this consideration: colony size appears as a good predictor of colony choice (Péron et al.2009). 2 &3). Tables Table 1: Proportion of failed (0), low-success (1), and high-success (2) colonies across four ranges of colony size. The dataset is constituted of 527 records of colony success for 31 years and 10 colonies ranging from 56 to 5000 pairs. Colony sizes are log-transformed.

Zusammenfassung

Figures

Size

  einer Lachmöwen-Population wurden die Effekte der Koloniegröße auf den individuellen Reproduktionserfolg untersucht. Die Koloniegröße lag bei 10 bis 5000 Paaren. Bezogen auf die Familiengröße (die Anzahl der betreuten Küken durch individuell markierte Eltern) und unter Berücksichtigung der interindividuellen Variation, wurde ein negativer Effekt der Koloniegröße während der ersten Lebenstage der Küken nachgewiesen. Dieser wurde kompensiert durch einen nachfolgenden Anstieg im Anteil der überlebenden Küken bezogen auf die Koloniegröße. Als Grund dafür vermuten wir die Wechselwirkung zwischen einem erhöhten Aufwand für den Schlupferfolg und die Vorteile einer Koloniebrut sowie einer möglicherweise effizienteren Nahrungssuche (Intensivierung der Nahrungssuche) für das Überleben der Küken. Allerdings erhöht sich die Häufigkeit vollständiger Koloniemisserfolge bei abnehmender Koloniegröße. Die Einbeziehung diese Risikos liefert eine verbesserte Abschätzung des Effektes der Koloniegröße auf den Bruterfolg und zeigt, dass die größten Brutkolonien die produktivsten sind. Dieses Muster entspricht vorherigen Ergebnissen, wonach größere Kolonien attraktiver sind für sich ansiedelnde Brutvögel.

Figures' legend Fig. 1 :

 legend1 Figures' legend Fig. 1: Family size, chicks" age and colony size: raw data presentation (circle size

Fig. 2 :

 2 Fig. 2: Family size, chicks" age (in days) and colony size (in pairs): estimations of the

Fig. 3 :

 3 Fig. 3: Post hoc model of reproductive success RS taking into account the variation

Fig. 1

 1 Fig.1

  Fig.2

Table 3 :

 3 Final GLMM for colony failure probability.

		Random effects
		SD
	Year	0.40
	Residual	0.82
		Fixed effects
		Estimate	95% CI
	Colony size 1.07	0.86 ; 1.27

Acknowledgments

Thanks are due to all volunteers, students and researchers who contributed to the field work. We are grateful to land owners who gave access to their properties. We thank in particular J. Arquillère, Manager of La Ronze, for continuous support and help throughout this study. T. Boulinier kindly offered helpful comments on a first draft of this article.

We are grateful to P.H. Becker, E.V.M. Stienen and an anonymous reviewer for improving this article.

The dataset is constituted of 1255 records of family size from 966 parents, for 31 years and 10 colonies ranging from 56 to 5000 pairs. Chick"s age and colony sizes are log-transformed. SD refers to random effects" estimated standard deviation value. CI refers to fixed effects" confidence interval.


Romain Marteau
email: romain.marteau@u-bourgogne.fr

Vincent Moron

Nathalie Philippon

Bernard Fontaine

Multi-scale approach of the onset of the rainy season over Sudano-Sahelian belt: spatial coherence and potential predictability

The spatial coherence of boreal monsoon onset (July-September) over the western and central Sahel is studied through the analysis of daily rainfall records for 136 rain-gauges from 1950-2000. Onset of the rainy season has been defined using 3 definitions which rely on 3 overlapped spatial scales: (i) the regional scale, i.e. the northward ITCZ jump from Guinean to Soudano-Sahelian latitudes, (ii) the meso-scale related with the first occurrence of the main rainfall-generating phenomenon, that is squall line and (iii) the local-scale of the first rainfall recorded at the rain-gauge. Local and meso-scale onsets show a weak degree of instantaneous and inter-annual spatial coherence, meaning that onset is almost never simultaneously recorded across a regional network but also that its inter-annual variability is not in-phase across such area. In consequence, the seasonal predictability of the monsoon onset coming from planetary and zonal sea surface temperature variations is weak.