Michelle Rosenzwajg

Roberta Lorenzon

Patrice Cacoub

Hang Phuong Pham

Fabien Pitoiset

Karim El Soufi

Claire Ribet

Claude Bernard

Selim Aractingi

Beatrice Banneville

Laurent Beaugerie

Francis Berenbaum

Julien Champey

Olivier Chazouilleres

Christophe Corpechot

Bruno Fautrel

Arsène Mekinian

Elodie Regnier

David Saadoun

Joe-Elie Salem

Jérémie Sellam

Philippe Seksik

Anne Daguenel-Nguyen

Jéremie Mariau

Eric Vicaut

David Klatzmann
email: david.klatzmann@sorbonne-universite.fr

Josef S Smolen

Basic and translational research

ABsTrACT

Objective regulatory t cells (tregs) prevent autoimmunity and control inflammation. Consequently, any autoimmune or inflammatory disease reveals a treg insufficiency. As low-dose interleukin-2 (ld-IL2) expands and activates tregs, it has a broad therapeutic potential. Aim We aimed to assess this potential and select diseases for further clinical development by crossinvestigating the effects of ld-IL2 in a single clinical trial treating patients with 1 of 11 autoimmune diseases. Methods We performed a prospective, openlabel, phase I-IIa study in 46 patients with a mild to moderate form of either rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, psoriasis, Behcet's disease, granulomatosis with polyangiitis, takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis and sclerosing cholangitis. they all received ld-IL2 (1 million IU/day) for 5 days, followed by fortnightly injections for 6 months. patients were evaluated by deep immunomonitoring and clinical evaluation. results ld-IL2 was well tolerated whatever the disease and the concomitant treatments. thorough supervised and unsupervised immunomonitoring demonstrated specific treg expansion and activation in all patients, without effector t cell activation. Indication of potential clinical efficacy was observed. Conclusion the dose of IL-2 and treatment scheme used selectively activate and expand tregs and are safe across different diseases and concomitant treatments. this and preliminary indications of clinical efficacy should licence the launch of phase II efficacy trial of ld-IL2 in various autoimmune and inflammatory diseases. Trial registration number nCt01988506.

InTrOduCTIOn

Regulatory T cells (Tregs) prevent autoimmunity and control inflammation. [START_REF] Wing | regulatory t cells exert checks and balances on self tolerance and autoimmunity[END_REF] Consequently, any autoimmune or inflammatory disease denotes a Treg insufficiency. Low-dose interleukin-2 (ld-IL2) expands and activates Tregs, and so has a broad therapeutic potential. [START_REF] Klatzmann | the promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases[END_REF] This potential is further supported by the central role of IL-2/IL-2 receptor in autoimmune diseases (AIDs), as recently highlighted in a genetic meta-analysis, [START_REF] Yr | Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases[END_REF] and by IL-2 pleiotropic functions. [START_REF] Klatzmann | the promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases[END_REF] Indeed, robust data demonstrate that IL-2 expands Tregs and blocks the differentiation of CD4 naïve T cells into follicular helper or proinflammatory helper 17 (Th17) T cells. 4 5 Therefore, ld-IL2 can act on three distinct arms of the immune response involved in AID pathophysiology: cellular and humoral immune responses and inflammation. Likewise, ld-IL2 is now being investigated in various clinical settings. [START_REF] Saadoun | regulatory t-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Koreth | Interleukin-2 and regulatory t cells in graftversus-host disease[END_REF][START_REF] Hartemann | Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial[END_REF][START_REF] Castela | Effects of low-dose recombinant interleukin 2 to promote t-regulatory cells in alopecia areata[END_REF][START_REF] Aa | Ultra low-dose IL-2 for GVHd prophylaxis after allogeneic hematopoietic stem cell transplantation mediates expansion of regulatory t cells without diminishing antiviral and antileukemic activity[END_REF][START_REF] Koreth | Low-dose Interleukin-2 in the treatment of Autoimmune disease[END_REF][START_REF] Rosenzwajg | Low-dose interleukin-2 fosters a dose-dependent regulatory t cell tuned milieu in t1d patients[END_REF] open trials have already yielded promising signs of efficacy, such as in systemic lupus erythematosus (SLE). 13 14 However, ld-IL2 efficacy may be affected by different factors: (1) Tregs from all patients may not respond similarly to ld-IL2, as some diseases may carry some intrinsic deficit of the IL-2 activation pathway 15-18 ; (2) Treg efficacy might be limited by high levels of inflammation such as during flares; (3) the existence of Tregs with appropriate T cell receptor antigen specificity that could be mobilised for therapy for each disease context is unknown; and (4) the global effect of ld-IL2 may be affected by the fact that Treg-dependent suppression of immune responses and inflammation depends on numerous cells, molecules and pathways that are likely to be affected differently in various AIDs. Finally, although ld-IL2 activates Tregs at doses at least 20-fold lower than for activating other cell types, 12 19 20 IL2 can affect effector T cells (Teffs), natural killer cells, type 2 innate lymphoid cells and eosinophils in a (high) dose-dependent manner. [START_REF] Ito | Ultra-low dose interleukin-2 promotes immunemodulating function of regulatory t cells and natural killer cells in healthy volunteers[END_REF][START_REF] Liao | Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy[END_REF][START_REF] Van Gool | Interleukin-5-producing group 2 innate lymphoid cells control eosinophilia induced by interleukin-2 therapy[END_REF] Thus, it remains to be seen whether a common appropriate dose/scheme of administration of ld-IL2 can be applied to various AIDs.

To address these questions, we designed a clinical trial in which we treated similarly ld-IL2 patients with 1 of 11 selected AIDs chosen to represent diverse pathophysiological processes. All patients received the same treatment and were monitored similarly. The aim was to cross-analyse the biological and clinical effects of ld-IL2 in heterogeneous patients, such as to appreciate the universality of ld-IL2 effects, and select diseases for conducting further phase II trials. We report here the results of the cross-analysis of 46 treated patients.

MeTHOds study design and participants

TRANSREG is a multicentre, interventional open study comparing biochemical and clinical responses to the administration of ld-IL2 across 11 selected diseases ( ClinicalTrials. gov trial registration number, NCT01988506). The selected diseases were rheumatoid arthritis (RA), ankylosing spondylitis (AS), SLE, psoriasis, Behçet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease (CD), ulcerative colitis (UC), autoimmune hepatitis and sclerosing cholangitis. Patients were selected based on common and disease-specific exclusion and inclusion criteria (online supplementary table S1A and S1B). The main inclusion criteria were a documented diagnosis of at least one of the selected diseases of mild to moderate activity, and being on stable standard therapy for ≥2 months at the time of inclusion. The main exclusion criteria were having another severe or progressive autoimmune/inflammatory disease, haematological disorders, vital organ failure, cancer, and active HIV, Hepatitis B Virus (HBV) or Epstein-Barr Virus (EBV) infections (online supplementary table S1A).

For homogeneity and proper cross-analyses, we report here the results of the first 46 patients who have been treated with IL-2 as Aldesleukin (Proleukin 18 MIU, Novartis Pharma SAS, Rueil-Malmaison, France), before we switched to a different formulation of IL-2 (ILT-101, ILTOO Pharma, Paris, France). Indeed, the use of Proleukin necessitates a cumbersome preparation by a pharmacist to dilute the product and prepare syringes that have limited time span.

Treatment

We previously showed the dose relation between IL-2 and Treg activation/expansion. 8 12 We selected the dose and scheme of administration of IL-2 used in TRANSREG from these results and a mathematical modelling of the long-term effects of IL-2 administration. [START_REF] Churlaud | pharmacodynamics of regulatory t cells in mice and humans treated with low-dose IL-2[END_REF] Likewise, patients received 1 Million International Units (MIU)/day of IL-2 from day 1 to day 5 (the induction period), and then every 2 weeks from day 15 to day 180 (the maintenance period). A follow-up visit was made 2 months after the end of the IL-2 treatment (day 240). Patients continued to receive their standard background therapy.

Immunomonitoring

All the immunomonitoring procedures are described in the online supplementary methods.

endpoints

The primary endpoint was the change in the relative concentration of peripheral blood Tregs on day 8 compared with baseline.

Biological secondary endpoints were the area under the curve (AUC) of the changes from baseline in relative concentration of Tregs during the maintenance period from day 30 to day 183 and the changes in inflammation markers from baseline to the end of treatment.

Clinical secondary endpoints were Clinical Global Impression (CGI), [START_REF] Busner | targum Sd. the clinical global impressions scale[END_REF] disease-specific and Five-level EuroQol Five-dimensional (EuroQL-5D-5L) scores. Chronic fatigue and arthralgia were also evaluated by physicians at baseline, month 3 and month 6 as they are the most common symptoms shared by patients across all pathologies included in this trial. All clinical evaluation procedures are described in the online supplementary methods.

statistical analysis

Changes in Tregs, immunological parameters and inflammation biomarkers between day 1 and day 8 were analysed using analysis of variance for ranked data (Conover's method) considering factor time and disease. The global effect of treatment at the initiation of treatment and its persistence during the maintenance phase was evidenced by demonstrating that the AUCs (calculated by the trapezoidal method) of the changes from baseline between day 1 and day 15 (iAUC D1-D15 ) and between day 30 and day 180 (mAUC D30-D180 ) were significantly different from zero using Wilcoxon test.

Changes in specific clinical scores and EuroQL-5D-5L were analysed by means of the Wilcoxon test. Changes in CGI severity and activity were analysed by t-test. Fatigue and arthralgia at months 3 and 6 were compared with baseline using Fisher's test.

resulTs

Fifty-one patients were included between January 2014 and May 2016. Five patients were not eligible, three because of a viral load of EBV greater than that permitted and two for intercurrent diseases. Thus, 46 patients were treated (figure 1). Patients were heterogeneous in terms of age (23-75 years), disease duration (10-536 months), body mass index (18.3-40.8), per cent of Tregs at baseline (2.2%-12.8%) and background therapy (online supplementary table S2). Several patients had other concomitant autoimmune or allergic diseases (online supplementary table S2). Concomitant Sjogren's syndrome (n=3), antiphospholipid antibody syndrome (n=3), morphea (n=1), Raynaud's phenomenon (n=1) or psoriasis (n=1) were observed in patients with SLE and RA. Concomitant allergic rhinitis (n=1), allergic asthma (n=1), multiple food allergy (n=1) and cutaneous contact hypersensitivity (n=1) were observed in patients with UC and psoriasis. In agreement with the inclusion criteria, the value of CGI activity and severity score ranged from 0 to 4 at baseline, with mild to moderate specific clinical scores for each disease. There were no other noticeable aspects of baseline demographic and laboratory characteristics of patients. No major deviations were observed during the study. The most common minor protocol deviations were out of windows visits (n=11) or drug administration not performed because of intercurrent diseases (n=7) in the maintenance period. Ten patients dropped out of the study (online supplementary table S3).

The mean±SD baseline percentage of Tregs in patients was 5.8%±2.3% of CD4 + T cells (online supplementary table S2). On day 8, the primary efficacy endpoint was reached with an increase of Tregs to a mean of 11.1%±4.6%, corresponding to a 2.0±0.6-fold increase (p<0.0001) (figure 2A and online supplementary table S4A). Treg expansion on day 8 appeared similar across the various diseases for which a minimum of four patients were treated (figure 2B), and cross-comparisons of these responses between diseases showed no significant differences (online supplementary table S4B). Moreover, we did not observe difference in Treg increase between patients receiving antiproliferative drugs and patients receiving non-steroid anti-inflammatory treatments or corticosteroids (online supplementary figure S1). On day 15, that is, 10 days after the last ld-IL2 administration of the induction phase, Treg increase was still significant (p=0.02) (figure 2A and online supplementary table S4A). Treg expansion persisted during the maintenance period. From months 1-6, the mean AUC value of Treg changes from baseline for all patients was significantly different from 0 (AUC M1- M6 =35.1±13.1, p<0.001). It is noteworthy that the recorded results may underestimate the true effect of ld-IL2 because Treg measurements were performed just before the IL-2 injections and thus capture only the residual increase from the previous injection 14 days earlier. The significant changes in percentages of Tregs were also observed for absolute numbers of Tregs (online supplementary table S4A).

There were no detectable effects of ld-IL2 on Teffs (defined as all Foxp3 -CD4 + and CD8 + cells), or on activated CD4 + C-D25 lo/+ Foxp3 -Teffs (online supplementary figure S2). This led to a 2.17±0.72-fold increase of the Treg:Teff ratio (p<0.0001) (figure 2C), as well as to a 1.98±0.42-fold increase of Tregs/ activated non-Treg CD4 + cells (p<0.0001) at the end of the induction course (figure 2D). It is worth noting that every single patient responded to 1 MIU/day of IL-2 by expanding their percentage of Tregs in the peripheral blood by at least 25%.

Because unwanted stimulation of Teff could be deleterious in the treatment of AIDs, the demonstration of a specific effect of ld-IL2 on Tregs is of utmost importance for the treatment of AIDs caused by Teffs. Although classic immunophenotyping did not show any expansion of non-Treg T cells, we further assessed that the effects of IL-2 are Treg-specific in a group of nine patients in whom an extensive immunophenotyping was performed [START_REF] Pitoiset | deep phenotyping of immune cell populations by optimized and standardized flow cytometry analyses[END_REF] and using unsupervised analyses. Using t-distributed stochastic neighbor embedding (t-SNE) and flowMeans R packages, [START_REF] Aghaeepour N | FlowMeans: non-parametric flow cytometry data gating. r package Version[END_REF][START_REF] Der Mlvan | Visualizing data using t-SnE[END_REF][START_REF] Van Der Maaten | Accelerating t-SnE using tree-based algorithms[END_REF] clusters were defined automatically based on Foxp3 and CD127 expression (figure 3A). Cluster 6 corresponded to Foxp3 + CD127 -/low cells and thus defines the Treg cluster, while the Teff cells corresponded to all the other clusters. We repeated the procedure to automatically recluster separately Tregs and Teffs based on the other markers of staining (CD25, Helios, CD45RA and CCR5). This generated six and eight clusters for Tregs and Teffs, respectively (figure 3B,D). For each cluster, comparison of the different values per marker on day 8 versus baseline showed statistically different values only for Tregs (p=0.03), and among them those with the more activated phenotypes (figure 3C). There were no differences for any of the Teff subsets (figure 3E), with p value not even close to significance. The same analysis strategy was then applied to another panel of antibodies (online supplementary figure S3). Here again, only two Treg subsets corresponding to those with the phenotype of either resting/naïve CD45RA + CD95 + CCR7 + ICOS +/-HLA-DR -) or activated/memory Tregs (CD45RA -C-D95 + CCR7 +/-ICOS +/-HLA-DR + ) were significantly expanded on day 8 while other T cell subsets were not affected.

Analysis of T, B and natural killer (NK) cell subsets showed little change during the induction period (online supplementary table S4A). As previously described, [START_REF] Rosenzwajg | Low-dose interleukin-2 fosters a dose-dependent regulatory t cell tuned milieu in t1d patients[END_REF] we observed an increased frequency of the regulatory CD56 bright NK cell subset (online supplementary figure S4A-C). Eosinophils levels were heterogeneous at baseline. For the 43 patients with normal counts, we observed a slight increase on day 8 that stayed under the normal value limit, or slightly above in two patients. For the three patients with eosinophilia at baseline, eosinophil counts approximately doubled after the induction period for two of them and remained stable for the other. All values returned to baseline during the maintenance treatment (online supplementary figure S5).

Plasma levels of Th1/Th2/Th17/Treg cytokines were unchanged all along treatment (online supplementary figure S6). In agreement with the mild to moderate activity of the diseases, C-reactive protein (CRP) was detected (>5 mg/dL) in only 10 patients at baseline and thus could not be studied as an endpoint (online supplementary figure S7).

We reported that ld-IL2 has a global anti-inflammatory effect based on a transcriptome analysis of peripheral blood mononuclear cells (PBMCs). [START_REF] Saadoun | regulatory t-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF] Here, we similarly analysed the global transcriptome of PBMCs before and after ld-IL2. We found 91 differentially expressed genes with a Benjamini-Hochberg corrected p value <0.05 on day 8 versus baseline (online supplementary table S5). Ingenuity Pathway Analysis showed significant enrichment of four pathways/signatures all directly or indirectly related to Treg. The most significantly modulated pathway (-log(p value)>3) is organised around upregulated genes such as Foxp3, IL-2Ra and Rb and CTLA4 that are essential for Treg function (figure 3F). We also assessed the modulation of a recent Treg signature defined from single cell transcriptomic. [START_REF] De Simone | transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating t regulatory cells[END_REF] This signature was significantly upregulated on day 8 versus baseline (p<1e-04) as well as at month 3 versus baseline (p<0.03), indicating that ld-IL2 effects on Tregs were maintained over time across diseases (online supplementary figure S8).

As previously reported, we did not observe anti-IL-2 antibodies in patients' plasma after ld-IL2 treatment (online supplementary figure S9). 12 31 Low-dose recombinant human (rh)IL-2 was well tolerated. Six patients displayed seven serious adverse events, none of which was considered related to IL-2 (online supplementary table S6A). Most non-serious adverse events (NSAEs) were injection site reactions, which occurred in approximately a quarter of the injections. The frequency of seasonal upper or lower respiratory tract infections (n=28), with associated fever of over 38°C in 17 of them, was as expected. The investigators did not report any unforeseen outcome of these infections (online supplementary table S6A). Finally, the analysis of the NSAEs according to background therapy did not show any significant difference (online supplementary table S6B).

Clinical secondary endpoints were CGI, disease-specific and EuroQL-5D-5L scores. CGI was selected as a clinical evaluation method that could work across our heterogeneous group of diseases. Indeed, CGI was originally developed for use in clinical trials to provide a brief, stand-alone assessment of the clinician's view of the patient's global functioning prior to and after initiating a study medication. [START_REF] Busner | targum Sd. the clinical global impressions scale[END_REF] CGI is commonly used in psychiatry but has not yet been validated in AIDs. Compared with baseline, a statistically significant improvement of the CGI scored by the physician was found at months 3 and 6 for CGI activity (p<0.001) (figure 4A) and at month 6 for CGI severity (p<0.001) (figure 4B). Significant changes were also found at the follow-up visit 2 months after discontinuation of the treatment (CGI activity p=0.02 and CGI severity p=0.04). Among the 46 treated patients, there were 26 with documented arthralgia and 26 with chronic fatigue at baseline. At month 3, there was a significant decrease of the percentage of patients with fatigue (p=0.002) and with arthralgia (p=0.00015) (figure 4C), and this trend continued at month 6. Evaluation of the impact of ld-IL2 on quality of life using EuroQL-5D-5L showed a non-significant improvement. We also assessed the disease-specific score for diseases with at least four patients treated. There was an improvement for AS (figure 5A), UC (figure 5B), SLE (figure 5C and online supplementary figure S9) and psoriasis (figure 5D,E), but not for CD (online supplementary table S7).

dIsCussIOn

We previously reported a double-blind, placebo-controlled dosefinding study of ld-IL2 in type 1 diabetes. [START_REF] Hartemann | Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial[END_REF] This identified a dose of 1 MIU/injection as well tolerated and boosting Tregs without effects on Teffs. 8 12 Given the heterogeneity of ld-IL2 target diseases and of their pathophysiological background, it remained to investigate how 'universal' would be the effects of this dosage. We thus initiated a trial aimed at cross-evaluating ld-IL2 in 11 different AIDs chosen to cover diseases that are organ-specific and systemic, T cell-mediated or antibody-mediated, and with high or little inflammation.

Our supervised analyses clearly demonstrated that 1 MIU/injection, with the scheme used, selectively activates and expands Tregs without activating Teffs, whatever the disease. This translates in a significant increase of both the Treg/Teff as well as the Treg/activated CD4+ T cell ratios. A similar response was observed for patients with low or high Treg counts at baseline. Both naïve and activated/memory Tregs expanded after ld-IL2, while we did not detect expansion of CD4 effector memory cells. As previously reported, at the dose used, we did not observe an effect on the overall NK cell population, but only on the non-cytotoxic CD56 hi NK cell subset, also called regulatory NK cells. Given the importance of the specificity of the effect on Tregs, we also evaluated it using unsupervised analyses that can be considered as less biased. This fully confirmed the specificity of the ld-IL2 effects for Tregs, further showing that the only cells responding to ld-IL2 were the resting/naive and activated/memory Tregs. Thus, supervised and unsupervised cellular and molecular analyses indicate that, with the dose/scheme used, ld-IL2 triggers a 'universal' specific effect for Tregs across a group of very heterogeneous patients.

Although this trial was powered only to evaluate the effects of IL-2 on Tregs, we also monitored secondary efficacy criteria relating to clinical status. With the idea of a cross-analysis of

Basic and translational research

diseases with various symptoms and scores, we chose CGI as our main per protocol clinical endpoint for cross-evaluation. Despite the fact that patients had mild or moderate disease forms, thus low CGI scores at baseline, and were heterogeneous, we observed an overall significant improvement of CGI scores. Improvements in CGI scores were already noted at month 3 and continued to increase at month 6. A potential clinical benefit was also evaluated across diseases by specifically monitoring arthralgia and chronic fatigue, which were the most shared symptoms of our patients.

There was a significant improvement of these symptoms, already noted at month 3. Two months after treatment discontinuation, CGI scores had a tendency to increase but were still significantly improved compared with baseline. No flare was observed during this period. Finally, we evaluated the disease-specific scores for diseases with such available scores and with at least four patients included. As previously reported, we saw improvement in SLE.

We also saw improvements for patients with UC, AS and psoriasis but not in those with CD. Altogether, these evaluations converge to suggest a broad potential of ld-IL2 and contributed to the selection of SLE as the target disease of an ongoing phase II trial (NCT02955615). They also suggest to further evaluate CGI in the field of AIDs. Indeed, as many drugs being developed target pathways involved in multiple AIDs, they thus have a broad therapeutic potential. Our trial design and methods could represent an early clinical cross-evaluation step that would help select diseases for further evaluation.

The safety profile of ld-IL2 across the different diseases and across various background treatments was very good. There has been no serious adverse event related to treatment. The most frequent adverse events were reaction at the injection sites, which are common for biologics, and are of unknown mechanisms. As in other trials of ld-IL2, we did not observe induction of anti-IL-2 antibodies under treatment. 12 31 Based on safety data from our previous clinical trials and our modelling of the effects of IL-2 on Tregs, [START_REF] Churlaud | pharmacodynamics of regulatory t cells in mice and humans treated with low-dose IL-2[END_REF] we used in this trial the dose of 1 MIU/injection, with once-a-fortnight injections during the maintenance course. Should a more pronounced effect on Treg during the maintenance phase be desired, one could use weekly injections as we do in our current LUPIL-2 trial (NCT02955615).

Altogether, our study highlights the 'universal' safety, biological efficacy and possible clinical efficacy of ld-IL2 across a group of very heterogeneous patients. It also highlights that the therapeutic window of plain IL-2 is satisfactory and thus licences the initiation of phase II efficacy trials, which are now necessary to ascertain the therapeutic potential of ld-IL2.

Figure 1

 1 Figure 1 (A) Trial profile. We included 51 patients suffering from 11 autoimmune diseases: rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic lupus erythematosus (SLE), psoriasis (P), Behcet's disease (BD), granulomatosis with polyangiitis (GWP), Takayasu's disease (TD), Crohn's disease (CD), ulcerative colitis (UC), autoimmune hepatitis (AIH) and sclerosing cholangitis (SC). Five patients were not eligible: three patients for an EBV viral load >1000 copies/mL and two patients for intercurrent diseases; 10 patients dropped out of the study (n in small inserts). (B) Study design. Patients received 1 MIU/day of interleukin-2 from D1 to D5 (the induction period), and then every 2 weeks from D15 to D180 (the maintenance period). D1, day 1; D5, day 5; D15, day 15; D30, day 30; EBV, Epstein-Barr Virus; M3, month 3; M6, month 6; M8, month 8; MIU, Million Intenational Units; V, visit.

Figure 2

 2 Figure 2 Changes in Treg cells and Teffs cells in patients treated with ld-IL2. Treg cells were gated in CD4 + T cells and identified as CD25 hi CD127 lo/-Foxp3 + cells. (A) Data represent changes in Tregs as percentages among CD4 + T cells for all patients from day 1 to month 8. (B) Data represent changes in Tregs in patients for diseases with n≥4. (C) Changes in Treg:Teff ratio defined as the percentage of Tregs divided by the percentage of the non-Treg CD4 + and CD8 + T cells. (D) Changes in Treg:activated CD4 + T cells ratio as the percentage of Tregs divided by the percentage of CD4 + CD25 lo/+ Foxp3 -T cells. (A-D) Data are represented as mean±SD. Data were normalised by baseline values for each patient at the different time points and are represented as fold change, but all statistics were made on raw data. Comparison between day 8 and baseline (main endpoint) was made by Wilcoxon signed-rank test. For each type of cell, global effect of the treatment at its initiation and its persistence during the maintenance phase was evidenced by showing that the AUC of the changes from baseline between day 1 and day 15 (iAUC D1-D15 ) and between day 30 and day 180 (mAUC D30-D180 ) was significantly different from 0 using Wilcoxon test. *P<0.05, **p<0.01, ***p<0.001. AS, ankylosing spondylitis; AUC, area under the curve; CD, Crohn's disease; D1, day 1; D8, day 8; D15, day 15; ld-IL2, low-dose interleukin-2; M1, month 1; M3, month 3; M6, month 6; M8, month 8; PSO, psoriasis; RA, rheumatoid arthritis; SC, sclerosing cholangitis; SLE, systemic lupus erythematosus; Teffs, effector T cells; Tregs, regulatory T cells; UC, ulcerative colitis.

Figure 3 Figure 4

 34 Figure 3 Unsupervised analysis of Treg phenotype and transcriptome. (A-E) Cells were stained for Helios, CD25, CXCR5, Ki67, CTLA-4, FOXP3, CD8, CD127, CD4 and CD8. A first step of automatic clustering based on the expression of FOXP3 and CD127 in CD3 + CD4 + cells was performed on a merge of samples from baseline and day 8 for the nine patients studied. (A) The algorithm generated seven clusters that are represented in different colours on a FoxP3/CD127 biplot. (B and D) A second step of automatic clustering was performed separately on Tregs (cells from cluster 6 in A) and Teffs (cells from all other clusters in A) based on FoxP3 -CD3 + CD4 + cells. (C and E) P values of the statistical analysis of the difference between baseline and day 8 for each cluster identified in B and D, respectively (statistical test: Wilcoxon pairwise test, p values <0.05 were considered as significant). (C) Also shows the phenotype of each cluster of Treg cells. (F) Transcriptomic analysis of significantly regulated genes from PBMCs on day 8 compared with baseline using Ingenuity Pathway Analysis reveals a Treg-related pathway (upregulated genes in red and downregulated genes in green; direct and indirect interactions between molecules are depicted by solid and dotted lines, respectively). PBMCs, peripheral blood mononuclear cells; Teffs, effector T cells; Tregs, regulatory T cells. on 27 November 2018 by guest. Protected by copyright. http://ard.bmj.com/ Ann Rheum Dis: first published as 10.1136/annrheumdis-2018-214229 on 24 November 2018. Downloaded from

on 27 Figure 5

 275 Figure 5 Clinical effects of low-dose interleukin-2 in specific diseases. Specific clinical scores were measured at baseline (day 1), month 3 (day 85), month 6 (day 183) and follow-up visit at month 8 (day 240): (A) BASDAI for patients with ankylosing spondylitis (n=10); (B) Mayo for patients with ulcerative colitis (n=4); (C) SLEDAI for patients with systemic lupus erythematosus (n=6); (D) BSA and (E) PASI for patients with psoriasis (n=4). Data are represented as means. BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BSA, body surface area; PASI,Psoriasis Area Severity Index; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index.

  on 27 November 2018 by guest. Protected by copyright. http://ard.bmj.com/ Ann Rheum Dis: first published as 10.1136/annrheumdis-2018-214229 on 24 November 2018. Downloaded from

  -Biotherapy department (i2B), Ap-Hp, pitié-Salpêtrière Hospital, paris, France 2 Immunology-Immunopathology-Immunotherapy (i3), Sorbonne Université, InSErM, paris, France 3 department of Internal Medicine and Clinical Immunology, Ap-Hp, pitié-Salpêtrière Hospital, paris, France 4 ILtoo pharma, paris, France 5 department of dermatology, Ap-Hp Cochin Hospital, paris, France 6 department of rheumatology, Ap-Hp pitié-Salpêtrière Hospital, paris, France 7 department of Gastroenterology, Ap-Hp Saint Antoine Hospital, paris, France 8 department of rheumatology, Ap-Hp Saint-Antoine Hospital, paris, France 9 department of Hepatology, Ap-Hp Saint-Antoine Hospital, paris, France 10 department of Internal Medicine, Ap-Hp, Saint-Antoine Hospital, paris, France 11 department of pharmacology and CIC-1421, Ap-Hp pitié-Salpêtrière Hospital, paris, France 12 department of pharmacy, Ap-Hp Saint-Antoine Hospital, paris, France 13 Unité de recherche Clinique and Université paris, Ap-Hp Saint Louis/Lariboisière Hospitals, paris, France

Rosenzwajg M, et al. Ann Rheum Dis 2018;0:1-9. doi:10.1136/annrheumdis-2018-214229

Acknowledgements

We thank the patients and the members of the trAnSrEG study group for their participation. We thank all the personnel of the Clinical Investigation Center paris-Est (CIC-pE), in particular C Funck-Brentano, E dasque, L Koehl and M Chenier, and the personnel of the Clinical Investigation Center for Biotherapies (CIC-Bti), Guillaume Churlaud, Michèle Barbié, Cornélia degbé, natalie Féry, Anne Laure raveu and Alexandra roux, for their excellent help. We thank G Bensimon for his initial contribution to the trial design and the selection of diseases. We also thank the UMr 8199 LIGAn-pM Genomics platform (Lille, France), which belongs to the 'Federation de recherche' 3508 LabEx EGId (European Genomic Institute for diabetes; Anr-10-LABx-46) and was supported by the Anr Equipex 2010 session (Anr-10-EQpx-07-01; 'LIGAn-pM'). the LIGAn-pM Genomics platform (Lille, France) is also supported by the FEdEr and the region nord-pas-de-Calais-picardie.

Contributors

Mr participated in the design of the study, supervised immunomonitoring, analysed the results and helped write the article. rL participated in the design of the study, was the principal clinical investigator, analysed the results and helped write the article. pC participated in the design of the study and was a clinical investigator of the study. Fp, KES and Hpp analysed the immunological and transcriptomic results. SA, BB, LB, FB, BF, JC, oC, CC, CF, AM, Er, dS, pS, JS and J-ES were clinical investigators of the study. Cr was an assistant clinical investigator and supervised the logistics. CB and Ad-n were in charge of treatment management. Vd and JM contributed to the study performance and regulatory affair follow-up. EV participated in the design of the study, wrote the statistical report, analysed the results and helped write the article. dK conceived and supervised the study, was the principal investigator, analysed the results and wrote the first draft of the article. All authors edited and approved the final draft of the article.

Funding Funding was provided by the sponsor, the Assistance publique -Hôpitaux de paris. ILtoo pharma provided support to the sponsor for clinical trial conduct and regulatory affairs, and contributed to some statistical analyses. Additional funding came from the Anr within the Investissements d'Avenir programme (Anr-11-IdEx-0004-02, LabEx transimmunom), the recherche Hospitalo-Universitaire (Anr-16-rHUS-0001, rHU IMAp) and the European research Council Advanced Grant (Fp7-IdEAS-ErC-322856, tripod).

Competing interests Mr, rL, pC, FB, BF, pC, JS, dS, CB and dK are inventors for patent applications related to the therapeutic use of ld-IL2, which belongs to their academic institutions and has been licensed to ILtoo pharma. Mr, Vd, JM and dK hold shares in ILtoo pharma. Hpp, Vd and JM are employees of ILtoo pharma. no other potential conflicts of interest relevant to this article were reported.

Patient consent obtained.

ethics approval the study was approved by the institutional review board of pitié-Salpêtrière Hospital (Cpp-IdFVI) and performed in accordance with the declaration of Helsinki and good clinical practice.

Provenance and peer review not commissioned; externally peer reviewed.


Déborah Iglicki
email: deborah.iglicki@univ-rennes1.fr

Mouktar Clément Goubault
email: clement.goubault@gmail.com

Nour Mahamoud

Soizic Chevance
email: soizic.chevance@univ-rennes1.fr

Fabienne Gauffre
email: fabienne.gauffre@univ-rennes1.fr

Shedding Light on the Formation and Stability of Mesostructures in Ternary "Ouzo" Mixtures

Keywords: Emulsion, Nanoprecipitation, Ouzo effect, Phase diagram, metastability

Hypothesis: Ternary systems made of water, a water-miscible solvent, and hydrophobic solutes spontaneously produce metastable particles by the "Ouzo effect" and thermodynamically stable "Surfactant Free Micro Emulsions" (SFME). However, the use of different analyses has led to a variability in the criteria to determine the boundaries of the Ouzo domain. We hypothesized that this could be clarified by investigating the stability and the physical state of the particles. Experiments: We investigate four systems using both solid and liquid solutes and two different solvents, and achieved a careful investigation of their phase diagrams, using DLS, Nanoparticle Tracking Analysis, NMR, Multiple Light Scattering, electrophoretic mobility, and fluorescence analysis. Findings: Our results evidence that the transition from the monophasic to the Ouzo domains does not coincide with the cloudiness curve, and that compositions in the Ouzo domain can look fully transparent, in contrast to what is often considered. This transition is best determined by stability analysis. The cloudiness curve corresponds to the formation of particles with a large size dispersity. In the Ouzo domain, we observed an exchange of solute between the continuous phase and solute particles swollen with solvent. In addition, the particles are stabilized against coalescence by their high negative charge.

1.Introduction

The role of the "Ouzo effect" in the elaboration of anis-flavored alcoholic beverage has long been recognized [START_REF] Vitale | Liquid Droplet Dispersions Formed by Homogeneous Liquid-Liquid Nucleation: "The Ouzo Effect[END_REF][START_REF] Grillo | Small-angle neutron scattering study of a world-wide known emulsion: Le Pastis[END_REF][START_REF] Chiappisi | Looking into Limoncello: The Structure of the Italian Liquor Revealed by Small-Angle Neutron Scattering[END_REF][START_REF] Scholten | The Life of an Anise-Flavored Alcoholic Beverage: Does Its Stability Cloud or Confirm Theory?[END_REF]. This phenomenon, more generally known as "nanoprecipitation" in the context of galenic and pharmaceutical sciences, is also widely used to elaborate polymer, drug or dye particles in the industry [START_REF] Lepeltier | Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices[END_REF][START_REF] Martínez Rivas | Nanoprecipitation process: From encapsulation to drug delivery[END_REF][START_REF] Ganachaud | Nanoparticles and Nanocapsules Created Using the Ouzo Effect: Spontaneous Emulsification as an Alternative to Ultrasonic and High-Shear Devices[END_REF]. More recently, it has been shown that Ouzo emulsions can also template the formation of capsules from polymers, proteins or nanoparticles [START_REF] Ganachaud | Nanoparticles and Nanocapsules Created Using the Ouzo Effect: Spontaneous Emulsification as an Alternative to Ultrasonic and High-Shear Devices[END_REF][START_REF] Yan | Simple but Precise Engineering of Functional Nanocapsules through Nanoprecipitation[END_REF][START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Goubault | The Ouzo effect: A tool to elaborate high-payload nanocapsules[END_REF][START_REF] Goubault | Radiosensitizing Fe-Au nanocapsules (hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy[END_REF]. The Ouzo emulsions/suspensions form in ternary systems consisting of two miscible solvents and a solute, this latter being highly miscible in one solvent but poorly in the other one. In most reported cases, the system contains water as one solvent, a polar water-miscible solvent (acetone, ethanol…) and a hydrophobic solute, such as an oil or a polymer [START_REF] Pucci | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF][START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF]. From a practical point of view, a fine emulsion is best obtained by a rapid mixing of water with the organic phase containing the solute [START_REF] Botet | How do interactions control droplet size during nanoprecipitation?[END_REF]. The solvent shift causes the instantaneous nucleation of solute-rich droplets that grow, fed by free solute molecules, following a "nucleation-and-growth" mechanism [START_REF] Vitale | Liquid Droplet Dispersions Formed by Homogeneous Liquid-Liquid Nucleation: "The Ouzo Effect[END_REF]. This first stage occurs rapidly (< 1s) and stops when the solute concentration in the continuous phase has decreased down to its saturation limit. A second period of droplet growth (t ~1h) is often described, either via coalescence or Ostwald ripening [START_REF] Lepeltier | Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices[END_REF][START_REF] Sitnikova | Spontaneously Formed trans -Anethol/Water/Alcohol Emulsions: Mechanism of Formation and Stability[END_REF][START_REF] Goubault | Effect of nanoparticles on spontaneous Ouzo emulsification[END_REF][START_REF] Roger | Coalescence of Repelling Colloidal Droplets: A Route to Monodisperse Populations[END_REF]. Interestingly, after this stage, droplet growth can be very slow and the colloidal stability of the samples can last for weeks. The existence of a charge on the surface of the droplets seems to be the most likely hypothesis to explain such stability. However, the origin of this charge is still a matter of debate [START_REF] Botet | How do interactions control droplet size during nanoprecipitation?[END_REF][START_REF] Ganachaud | Central Role of Bicarbonate Anions in Charging Water/Hydrophobic Interfaces[END_REF][START_REF] Roger | Surface Charge of Polymer Particles in Water: The Role of Ionic End-Groups[END_REF]. The final size of the droplets/particles ranges from ca 100 nm to a few microns, depending on the composition and preparation pathway.

One of the most attractive features of Ouzo emulsions is that very narrow size distributions can be obtained at specific compositions. In addition, the nucleation-and-growth mechanism is a spontaneous phenomenon allowing the "bottom up" generation of droplets/particles with almost no external energy [START_REF] Vitale | Liquid Droplet Dispersions Formed by Homogeneous Liquid-Liquid Nucleation: "The Ouzo Effect[END_REF][START_REF] Scholten | The Life of an Anise-Flavored Alcoholic Beverage: Does Its Stability Cloud or Confirm Theory?[END_REF]. This contrasts with conventional "top down" emulsification, where a high amount of energy is needed to fractionate and disperse one phase into the other.

In many cases, the formation of nanoparticles by nanoprecipitation/Ouzo effect is based on empirical experience and visual observation of cloudiness. However, a precise knowledge of the phase diagrams can greatly improve the quality of the size distribution and also enable to elaborate multicomponent particles [START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Goubault | The Ouzo effect: A tool to elaborate high-payload nanocapsules[END_REF]. The ternary phase diagrams of Ouzo systems generally show a biphasic domain, separated from the monophasic domain by the binodal curve [START_REF] Arce | Revising Concepts on Liquid-Liquid Extraction: Data Treatment and Data Reliability[END_REF]. The other important curve is the spinodal curve, that sets the system stability limit. The space enclosed in between the binodal and the spinodal curves corresponds to a metastable region. Note that the Ouzo domain is a sub-area of the metastable region, that it is not thermodynamically defined and depends on sample preparation conditions [START_REF] Pucci | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF][START_REF] Botet | How do interactions control droplet size during nanoprecipitation?[END_REF]. For instance, the boundary determined by step-by-step addition of water to the solute organic solution often differs from that obtained by addition of water in one shot [START_REF] Pucci | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF]. Very interestingly, thermodynamically stable microemulsions may be observed in the monophasic (transparent) domain [START_REF] Klossek | Eco-solvents -cluster-formation, surfactantless microemulsions and facilitated hydrotropy[END_REF]. These have been called "surfactant free microemulsion (SFME)" [START_REF] Hou | Surfactant-free microemulsions[END_REF] or "pre-Ouzo ultraflexible microemulsions" [START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Lucia | Formation of surfactant free microemulsions in the ternary system water/eugenol/ethanol[END_REF]. Their sizes vary from a few [START_REF] Zemb | How to explain microemulsions formed by solvent mixtures without conventional surfactants[END_REF][START_REF] Prévost | Spontaneous Ouzo Emulsions Coexist with Pre-Ouzo Ultraflexible Microemulsions[END_REF][START_REF] Diat | Octanol-rich and waterrich domains in dynamic equilibrium in the pre-ouzo region of ternary systems containing a hydrotrope[END_REF] to ca 100 [START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Goubault | Effect of nanoparticles on spontaneous Ouzo emulsification[END_REF] nanometers, which suggests that they might have a different origin.

Various techniques were used to investigate the structures formed in the Ouzo systems and determine the boundaries between the different domains. Among them Dynamic Light Scattering (DLS) is the most frequently reported, as it is a widespread technology that can measure the size of Brownian particles from a few nanometers to a few microns. Its main limitations are that it is not appropriate for cloudy samples (due to multiple light scattering) nor for polydisperse samples (for which it is difficult to obtain a proper distribution). Small angle X-Ray Scattering and Neutron Scattering (SAXS and SANS) also brought interesting insights but cannot be used routinely [START_REF] Grillo | Small-angle neutron scattering study of a world-wide known emulsion: Le Pastis[END_REF][START_REF] Chiappisi | Looking into Limoncello: The Structure of the Italian Liquor Revealed by Small-Angle Neutron Scattering[END_REF][START_REF] Zemb | How to explain microemulsions formed by solvent mixtures without conventional surfactants[END_REF][START_REF] Diat | Octanol-rich and waterrich domains in dynamic equilibrium in the pre-ouzo region of ternary systems containing a hydrotrope[END_REF]. A few studies have taken advantage of NMR to probe the size of the aggregates and the possible exchange of solute between the continuous phase and the aggregates [START_REF] Bassani | Probing the Initial Events in the Spontaneous Emulsification of trans -Anethole Using Dynamic NMR Spectroscopy[END_REF][START_REF] Tehrani-Bagha | An Ouzo emulsion of toluene in water characterized by NMR diffusometry and static multiple light scattering[END_REF]. Finally, electron microscopy is frequently used to image the particles obtained when working with solid solutes, particularly polymers [START_REF] Pucci | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF][START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF].

Until now, most authors have identified the limit of cloudiness with the boundary of the Ouzo domain. This originates from the fact that the concept of Ouzo emulsions was developed to explain the cloudiness of this beverage, but the reciprocal -all samples in Ouzo domain should be cloudy-is not necessarily true. More generally, it is a common mistake to identify the binodal with the cloudiness curve as recently pointed out by Arce et al [START_REF] Arce | Revising Concepts on Liquid-Liquid Extraction: Data Treatment and Data Reliability[END_REF].

In our group, we have used the water/THF/BHT (THF = Tetrahydrofuran, BHT = butylated hydroxytoluene) to elaborate capsules with a nanoparticle shell [START_REF] Goubault | Effect of nanoparticles on spontaneous Ouzo emulsification[END_REF][START_REF] Sciortino | Simple Engineering of Polymer-Nanoparticle Hybrid Nanocapsules[END_REF]. The choice of this system was driven by the fact that BHT, generally present in commercial THF as a radical scavenger, is known to generate submicronic droplets [START_REF] Li | Large-Scale Structures in Tetrahydrofuran-Water Mixture with a Trace Amount of Antioxidant Butylhydroxytoluene (BHT)[END_REF][START_REF] Bunkin | Droplet-like heterogeneity of aqueous tetrahydrofuran solutions at the submicrometer scale[END_REF]. In this system, we observed transparent samples containing droplets of ca 100 nm. These samples were classified as SFME as they did not seem to evolve with time [START_REF] Goubault | Effect of nanoparticles on spontaneous Ouzo emulsification[END_REF]. Interestingly, this suggests that samples in the Ouzo domain might also look transparent since we did observe a size continuity between both domains.

With the aim to find an objective method to determine the boundaries of the Ouzo domain we investigated in details the model system water/THF/BHT using Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Static Multiple Light Scattering (SMLS), Nuclear Magnetic Resonance (NMR) and electrophoretic mobility. In particular, we consider stability (i.e. no evolution of droplet size) as a criterion to distinguish between the SFME (stable) and the Ouzo (slow droplets growth) domains. Three new systems: water/THF/α-tocopherol, water/DMSO/limonene and water/THF/BODIPY are also considered.

Material and methods

Materials. Tetrahydrofuran (THF, inhibitor-free, HPLC grade, ≥99.9%) was purchased from Sigma-Aldrich. Butylated hydroxytoluene (BHT) was purchased from Janssen. (S)-4-Isopropenyl-1-methyl cyclohexene (S-(-)-limonene, 96%) was purchased from Sigma-Aldrich. (±)-α-Tocopherol (Vitamin E or Vit E) in oil solution from BASF was graciously provided by AMI-ingredient. 4-Difluoro-8-(4trimethylsilylethynylphenyl)-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) was synthesized following a reported procedure [START_REF] Ulrich | Convenient and Efficient Synthesis of Functionalized Oligopyridine Ligands Bearing Accessory Pyrromethene-BF 2 Fluorophores[END_REF]. Samples were made using MilliQ water (18.2 MΩ.cm).

Sample preparation and elaboration of the phase diagram.

In all cases, samples were prepared by adding water to a solvent (THF or DMSO) containing the hydrophobic solute (BHT, limonene, α-Tocopherol or BODIPY) in one shot. The water is added promptly using a micropipette and the sample is vortexed about 10 s unless stated otherwise. For elaborating the phase diagrams, the samples were immediately visually inspected under the light of a telephone flash. The limpid and homogeneous solutions were classified in the monophasic phase. If we observed a light scattering and a cloudiness inside the homogeneous samples, we attributed them to the Ouzo domain. If we saw any precipitates in the samples, or any oil droplets at the top of the solutions, we placed them in the diphasic domain. Concerning the elaboration of the BODIPY phase diagram in THF and water, the samples were made as previously explained and they were observed under UV illumination at λ excitation = 365 nm. Then we took a photography of each series at the different solvent fraction. And we placed a spot of the same color as the sample from these photos in the phase diagram at the coordinates corresponding to the different mass fractions of BODIPY and solvent used.

Static Multiple Light Scattering (SMLS).

A Static Multiple Light Scattering (SMLS) device (TurbiScan™ TOWER) from Formulaction (France) was used for monitoring the stability of the emulsions. The device measures Transmission (T, at 180°) or Backscattering (BS, at 45°) intensities over the sample height over time using a near infrared light source (λ = 880 nm) and two synchronous detectors that move up and down along the sample. The samples were prepared in glass vials by adding water in one shot to the organic solution. For mixing, each sample was slowly turned over 3 times and monitored by the TurbiScan™ instrument and the TowerSoft software for 14 h. Results are provided as the relative transmission.

Nanoparticle Tracking Analysis (NTA). NTA was carried out using a Nanosight LM10 device (Malvern Panalytical). The sample was inserted into the cell of the device and illuminated by a laser beam (40 mW, λ = 405 nm). The light scattered by the droplets appears as individual spots and recorded by a CCD camera, operating at 30 frames per second, through a microscope. For each sample, measurements were achieved a few minutes after mixing and after 3 h. Each measurement consists of 3 acquisitions of 30 s. Thanks to the NANOSIGHT NTA 2.0 Analytical Software, the size of each object was then individually deduced from the analysis of its Brownian motion. Measurement are operated at 25°C and calculations of the hydrodynamic diameters were achieved taking the viscosity of the THF/water mixture at this temperature from values reported by Das et al [START_REF] Mahendra | Densities and viscosities of the binary aqueous mixtures of tetrahydrofuran and 1,2-dimethoxyethane at 298[END_REF].

Dynamic Light Scattering (DLS). DLS measurements were achieved using a Vascokin DLS device from Cordouan (France) equipped with a contactless remote optical head measuring the scattered intensity from a laser diode (638 nm) at an angle of 170°. The scattered signal was acquired continuously during the desired time (1 to 3 h). Then, short sequences (typically of 1.5 to 3 min) were used at the selected time points for analysis. The hydrodynamic diameter (Z-average) is calculated by the method of the cumulants. Measurement temperature (room temperature) was monitored and input as a parameter in the cumulant fit.

Electrophoretic mobility. Electrophoretic mobility was measured using a Malvern Nanosizer operating at 633 nm and equipped with a dip cell (ZEN1002). Measurements were achieved right after mixing and after 15 min. The reported values represent the mean and standard deviation of three independent sets of 100 measurements.

Nuclear Magnetic Resonance (NMR).

All spectra were recorded using the PRISM core facility on a Bruker AVANCE NEO 500 MHz spectrometer equipped with a 5 mm TCI 1 H-13 C-15 N cryo-probe. 1 H spectra were recorded at 298 K using standard Bruker sequences. Different compositions of D 2 O/ THF-d8/BHT were analyzed by 1 H NMR. Briefly, the desired volume of BHT stock solution prepared in THF-d8 was diluted into the desired volume of THF-d8. Then D 2 O was quickly added and the solution was mixed 30 s by vortexing. NMR experiment was performed immediately after vortexing. The solution was then placed in a 5 mm NMR tube into which a coaxial tube was sometimes inserted but not systematically. This coaxial tube contained 200 µM of TSP (Trimethylsilyl)propionic-2,2,3,3-d 4 acid sodium salt) was used as external reference for calibration of the spectra and relative integration of the BHT signals. 2D 1 H NOESY experiments were carried out using the standard Bruker noesygpph sequence and the following acquisition parameters: mixing time of 500 ms, TD 4k x 128 points, 8 scans. This mixing time was long enough to detect chemical exchange. We used the freeware EXSYcalc (Mestrelab Research, https://mestrelab.com/software/freeware/) to estimate the kinetics constants k 1 and k -1 after integration of the off-diagonal cross-peaks. Self-diffusion measurements (DOSY experiments) [START_REF] Mahendra | Densities and viscosities of the binary aqueous mixtures of tetrahydrofuran and 1,2-dimethoxyethane at 298[END_REF] were carried out using a 2D Stimulated Echo experiment using bipolar gradients pulse sequence (stebpgp1s), with a diffusion delay (d20) of 350 ms and a gradient duration (p30) of 3 s. The diffusion dimension was processed with the 2D DOSY processing Topspin software (logarithmic scale).

Results and discussion

System water/THF/BHT. All samples in this study were prepared by addition of water to the organic solution in one shot, and no surfactant was used to stabilize the droplets. We first built a phase diagram water/THF/BHT by visual observations (see Figure SI-1 for a full diagram and Figure 1a for a zoom on the area of interest). We adopted the representation proposed by Vitale and Katz [START_REF] Vitale | Liquid Droplet Dispersions Formed by Homogeneous Liquid-Liquid Nucleation: "The Ouzo Effect[END_REF] where the mass fractions of solute and solvent are represented as horizontal and vertical axis respectively. The mass fraction of water is  Wat = 1-( THF + BHT ). Since the region of interest is located at very small mass fractions of BHT, the corresponding axis is represented on a Log scale. 2 × 10 -5 141.9 ± 1.4 248.1 ± 6.5 All samples were observed typically between 10 to 30 min after preparation. The full line represents the onset of cloudiness. At low THF fraction, the clouding is very subtle and difficult to detect by naked eyes. In this case, we used a cell phone light to shine the samples. At  THF > 0.2, the transition from transparent to cloudy compositions is more obvious and could be detected without a light. At high BHT concentrations, the samples rapidly form a thin turbid top layer, rich in oil (open triangles). We consider this limit (dotted line) as the spinodal line. The prediction of the binodal curves using semi-empirical methods to calculate the liquid-liquid equilibrium generally yield a straight line in the semi-Log representation [START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF]. Our own calculations of the binodal curve using COSMO-RS (Figure SI-1) also provide an almost straight line, however slightly shifted toward larger BHT mass fractions compared to the experimental data. Therefore, we assume that the linear portion of the cloudiness curve observed in the top part of the diagram is a good approximation of the binodal. The experimental curve clearly deviates from this expected linear behavior at mass fractions of THF lower than 0.2. In the following we investigate more specifically the samples series a n , b n and c n as represented in Figure 1a (see Table 1 for compositions).

Figure 1: (a) Phase diagram water/THF/BHT determined by visual observation and focused on the area of interest: (□) limpid samples; (•) cloudy samples; (Δ) biphasic samples. (──) cloudiness curve, (---) linear extrapolation of the cloudiness curve, (…..) boundary between cloudy and macroscopically biphasic samples. (b) Size distribution measured using NTA at time 0 (──) and 3 hrs (---) for the compositions a 2 -a 4 and c 0 -c 6 as indicated on graph a. (c) SMLS stability analysis: relative transmission measured at mid-height of the samples. Full line (──): limpid samples; dashed line (---): cloudy samples

a 7 3 × 10 -5 0.1 - - b 1 2.5 × 10 -6 - - b 2 7.4 × 10 -6 - - b 3 1 × 10 -5 - - b 4 1.6 × 10 -5 - - b 5 2 × 10
The composition line  THF = 0.2 (samples c n ) was selected for a first series of investigation because the location of the binodal curve is unambiguous. Samples of compositions ranging from  BHT = 2.5 10 -6 to 5 10 -5 (c 0 -c 6 ,) were analyzed using NTA ca 5 min after sample preparation, and also after 3 h to assess sample stability (Figure 1b). It is obvious that samples c 0 -c 3 , that look transparent, contain in fact particles of diameter in the range 60-80 nm and show no significant evolution within 3 hours. Beyond the cloudiness curve (c 5-7 ), the samples become more polydisperse and exhibit larger sizes (> 100 nm) that exceed the limitation of the NTA (1µm) after 3 hours. An example of this behavior is sample c 6, shown in Figure 1b. These samples were also examined using DLS (Figure SI-2). For the most diluted samples (c 0 -c 2 ), the scattered intensity is extremely low and a proper fit of the correlograms is prevented by the presence of a few very large particles (> 1µm). Figure SI-3 shows the effect of such large particles on the correlogram of composition c 1 , together with the visual observation of the corresponding scattering spots using NTA. In contrast, sample c 3 -c 6 could be analyzed. For compositions c 3 and beyond, we could achieve DLS analysis. Figure SI-2b shows the intensity-weighted size histogram for samples c 3 and c 6 shortly after mixing and after one hour. As expected, the mean size measured by DLS (225.6 ± 9.0 nm and 352.7 ± 11.4 nm for samples c 3 and c 6 at t= 0 respectively) is larger than those measured by NTA (95.2 ± 4.7 nm and 231.9 ± 13.8 nm respectively). In addition, the results confirm that c 3 does not evolve with time whereas the mean particle size increases over one hour for c 6 .

It is important to note that the Ouzo systems investigated in the literature are often stabilized by the addition of surfactant to prevent coalescence [START_REF] Ganachaud | Nanoparticles and Nanocapsules Created Using the Ouzo Effect: Spontaneous Emulsification as an Alternative to Ultrasonic and High-Shear Devices[END_REF][START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF]. Here, in the contrary, we assumed that monitoring the stability of the samples should be a method of choice to determine accurately the binodal curve. To this aim we used SMLS. The principle is to monitor the optical features (transmission and/or backscattering) during time and over the whole height of the sample. Coalescence or ripening is generally characterized by a similar evolution of the optical signal over the whole height of the sample, whereas sedimentation or creaming cause an uneven vertical optical signal. As an example, Figure SI-4 shows the time variation of the transmission (in relative values) for two samples on each side of the binodal curve: c 3 , fully transparent and c 5 , slightly cloudy. The transmission is measured all along the height of the vial. The kinetic behaviors of these two samples are strikingly different: sample c 3 remains stable over the 14 h of monitoring, whereas the transmission of sample c 5 clearly decreases with time, over the whole sample height. Very interestingly, the evolution of the sample reaches a stop after a few hours, but no sign of creaming or sedimentation was observed. This suggests that the mechanism(s) responsible for the growth of the droplets/particles is (are) hindered at some point. One hypothesis is that molecules (either free or under the form of very small aggregates) serving as a reservoir for the droplets are consumed [START_REF] Carteau | The "Ouzo effect": Following the spontaneous emulsification of trans-anethole in water by NMR[END_REF]. Alternatively, the coalescence of droplets can come to a halt when their surface is efficiently covered by stabilizers [START_REF] Goubault | Effect of nanoparticles on spontaneous Ouzo emulsification[END_REF]. The stability of samples c 1 -c 7 was investigated, and the time evolution of the transmission measured at mid-height is reported Figure 1c. Consistently with the previous results, compositions c 1 -c 3 are stable over the period of time considered, whereas c 5 -c 7 show a significant diminution of their transmittance. Thus, SMLS analysis shows a clear transition in the colloidal stability of the samples. NTA and DLS measurement operated at different times confirm the stability behavior of the samples but are less convenient to use in this respect.

Encouraged by these results, we monitored the stability of samples along the composition lines  THF =0.1 (samples a 1 -a 7 , Figure 1c) and  THF =0.15 (samples b 1 -b 5 , Figure SI-5). Samples a 0 and a 1 are stable, whereas samples a 2 -a 7 all evolve with time, with faster kinetics as BHT concentration increases. Similarly, compositions b 1 -b 2 are stable, and b 3 -b 5 unstable. Very interestingly, the samples a 2 -a 4 and b 3 look fully transparent, but are in fact unstable. This confirms the fact that the visual determination of the binodal line is not an efficient method. On the other hand, the transition between stable and unstable compositions is consistent with the extrapolated straight-line in Figure 1a, for both series a n and b n . We further analyzed compositions in the area in-between the cloudiness curve and the extrapolated dashed line (a 2 -a 4 ). Figure 1b shows their size distribution at initial time and after 3 hours. All three samples clearly contain a distribution of particles growing in size over time, which confirms that these compositions are in fact metastable. Note that because the Ouzo effect is considered as a spontaneous emulsification, some authors suggested it requires no mixing and mixing modes should have no effect on the final size distribution. However, this is not true. The nucleation conditions in the Ouzo domain are sensitive to mixing conditions [START_REF] Botet | How do interactions control droplet size during nanoprecipitation?[END_REF]. A rapid mixing rate (compared to nucleation rate) favors homogeneous nucleation. Therefore, we found it necessary to check if habits of different operators regarding mixing could influence our results, and found that it was not the case (see Supporting Information, Figure SI-6).

THF ( ). (B) 2D 1 H NOESY and (C) pseudo 2D 1 H DOSY NMR spectra of sample c 6 .

In order to follow the transition from the monophasic to the Ouzo domain in more details, especially in terms of both BHT environment and mobility, we recorded the 1D 1 H NMR spectra of samples c 2 -c 3 (SFME domain), c 4 (near the binodal) and c 6 (Ouzo domain). These spectra are presented in Figure 2A together with that of BHT solubilized in deuterated THF-d8. We can observe in spectra b-c a single set of BHT signals for the compositions c 2 and c 3 (SFME domain). In addition, the chemical shifts of the methyl protons (▲) are significantly moved toward high field. These observations are consistent with BHT mainly dissolved in the continuous phase made of water/THF, and possibly in rapid exchange with SFME droplets. In contrast, the compositions c 4 and c 6 show two sets of signals (full and empty symbols, spectra d-e), revealing that the BHT is distributed between two different environments, most probably the dispersed and continuous phase, with slow exchange at the chemical shift time scale. This strongly suggests that the droplets that appear past the binodal have a different composition from that in the SFME. In a previous study of the water/ethanol/trans-anethol system, a similar behavior was found, and the systematic deshielding effect for the 1 H resonances of form 1 compared to form 2, was attributed to its more hydrophilic environment [START_REF] Carteau | The "Ouzo effect": Following the spontaneous emulsification of trans-anethole in water by NMR[END_REF]. Moreover, the integration of the signals clearly shows that the proportion of BHT in this environment increases with the total amount of BHT: it represents > 60% of the BHT for the c 6 sample. To confirm the origin of these two sets of signals, we recorded the 2D 1 H NOESY (Nuclear Overhauser Effect SpectroscopY) spectrum of the sample c 6 (Figure 2B). This powerful experiment evidences, i) spatial correlations (nOes, distance < 5 Å) between two different protons (the green off-diagonal cross peaks in the Figure 5a) and ii) the presence of two species in chemical exchange (the blue off-diagonal cross peaks) [START_REF] Jeener | Investigation of exchange processes by two-dimensional NMR spectroscopy[END_REF][START_REF] Kaiser | Use of the Nuclear Overhauser Effect in the Analysis of High-Resolution Nuclear Magnetic Resonance Spectra[END_REF][START_REF] Anet | Nuclear Magnetic Resonance Spectral Assignments from Nuclear Overhauser Effects1[END_REF]. This latter observation confirms the presence of two liquid BHT species in exchange, one in the continuous phase and the other in the dispersed phase. The exchange constant kinetics were estimated at k 1 and k -1 at 0.23 s -1 and 0.16 s -1 respectively [START_REF] Frunzi | Kinetics and Solvent-Dependent Thermodynamics of Water Capture by a Fullerene-Based Hydrophobic Nanocavity[END_REF]. To our knowledge, it is the first time that this exchange dimension between the continuous and dispersed phases is so clearly demonstrated. Then, we recorded DOSY spectra that allow us to discriminate the chemical species contained in a mixture in solution based on their self-diffusion coefficient. As shown in Figure 2C, the two BHT species coexisting in solution for sample c 6 do not have the same self-diffusion coefficient. The BHT contained in the continuous phase diffuses faster (~ 5 10 -10 m².s -1 ) than the BHT in the droplets of the dispersed phase (~ 2 10 -10 m².s -1 ). We can explain this result by a significantly higher viscosity in the dispersed phase compared to the continuous phase, due to the high BHT concentration within the droplets. The NMR analysis of the sample a 6 (Figure SI-7) yields similar conclusions as for the sample of composition c 6 presented above: two liquid BHT species coexist in solution and exhibit different diffusion coefficient, ~ 6.6 10 -10 m².s -1 and ~ 1.6 10 -10 m².s -1 , corresponding respectively to the BHT in the continuous and dispersed phases. Note that the viscosity of the water/THF mixtures [START_REF] Mahendra | Densities and viscosities of the binary aqueous mixtures of tetrahydrofuran and 1,2-dimethoxyethane at 298[END_REF] is significantly lower for the composition a 6 (10% THF, = 1.2 cP) compared to composition c 6 (20% THF, = 1.5 cP), consistently with the higher diffusivity of the dissolved BHT in sample a 6 . The predominant species is the BHT in the dispersed phase and represent ~75% of the total BHT. Unfortunately, despite the use of a more sensitive cryo-probe, the analysis of samples with lower BHT concentration was not possible.

As mentioned earlier, the presence of an electrostatic charge that inhibits coalescence is the most probable explanation for the slow evolution of Ouzo droplets over the long term. Therefore, we examined the electrophoretic mobility of the aggregates along the composition lines  THF =0.1 and  THF =0.2 (Figure 3). The mass fractions of BHT were normalized by their value at the binodal curve (dashed curve in Figure 1a), respectively  * BHT = 3.5 10 -6 and  * BHT = 2 10 -5 . It is striking that for both curves, the electrophoretic mobility is negative and reaches a minimum at the transition from the SFME to the metastable domains. Since the pH of these mixtures (pH=7  0.5 and 6.5  0.5 for  THF =0.1 and 0.2 respectively) were relatively constant when varying the amount of BHT, this cannot be attributed to a change of concentration in OH -. The strongly negative values (close to -80 mV in terms of zeta potential) reached for  THF =0.1 explains the very slow evolution of compositions a 2 -a 6 that was revealed by SMLS (Figure 1c). It has long been known that the hydrophobic interfaces in water often carry a negative charge. Several hypotheses were proposed to explain this phenomenon, including the interfacial adsorption of endogenous negative ions such as water carbonates or hydroxides, or the presence of ionizable impurities [START_REF] Ganachaud | Central Role of Bicarbonate Anions in Charging Water/Hydrophobic Interfaces[END_REF][START_REF] Roger | Why Are Hydrophobic/Water Interfaces Negatively Charged?[END_REF]. Alternatively, some theoretical works assume that the surface charges from asymmetric charge density of water molecules in the vicinity of the interface [START_REF] Vácha | The Orientation and Charge of Water at the Hydrophobic Oil Droplet-Water Interface[END_REF]. Interestingly the coalescence of electrostatically repelling droplets can even lead to size focusing [START_REF] Roger | Coalescence of Repelling Colloidal Droplets: A Route to Monodisperse Populations[END_REF]. At the present time, we do not have a clear interpretation for the increase of surface charge close to the binodal. This could indicate a change in the droplet structure, with a sharper interface after the binodal than in the SFME. At this point, we have confirmed that a simple visual observation is not appropriate to determine the location of the binodal in the water/THF/BHT system at low solute concentrations. Monitoring the evolution of the sample through light scattering techniques such as SMLS, but also NTA or DLS enables to find the expected linear shape of the binodal curve in the standard semi-Log representation. In order to generalize these findings, we decided to investigate new systems.

Investigation of systems with an oil as solute. We considered the systems water/THF/α-tocopherol and water/DMSO/limonene. Both limonene and α-tocopherol (vitamin E) are oils. The phase diagrams were first built by visual classification of the samples between limpid, cloudy and biphasic samples. As previously, the cloudiness curve deviates from the binodal at low solvent fractions (Figure 4). SMLS analysis was performed for compositions of interest (Figure SI-8). For the system water/DMSO/limonene, the stability analysis confirms that the binodal curve corresponds to the straight line extrapolated from the high concentrations. Indeed, compositions above this curve are stable, whereas compositions below this curve evolve with time. In the case of water/THF/α-tocopherol, the variations of transmittance for samples around the binodal curve are too small to properly identify the transition. This is due to the fact that the mass fraction of α-tocopherol at the binodal is below 10 -6 , for the corresponding line. NTA analysis was also performed, and for each line, the mean hydrodynamic diameter is plotted as a function of the solute mass fraction normalized by the mass fraction at the cloudiness curve (full line, Figure 4). This representation clearly evidences, for all three systems, a sudden increase (typically by a factor 2) in the mean size of the droplets at the cloudiness curve. In contrast, the size remains almost constant over two decades of mass fractions below this curve. Beyond this limit, the size becomes rapidly too large and/or the distribution too wide to be determined by NTA. Interestingly, this result indicates that the cloudiness curve is not a simple limit of detection but has a physical meaning. Our understanding of this boundary is that on its left side exists a transparent Ouzo domain consisting of small droplets (typically ≤ 100nm) with low dispersity, and on its right side a cloudy Ouzo domain with large and/or polydisperse droplets. At this point, it is interesting to note that there is a size continuity between the SFME and Ouzo domains. A possible explanation is that SFME aggregates exist in the Ouzo domain close to the boundary, as a supersaturated situation. However, the Ouzo domain is ultimately bound to form two separate phases. Therefore, these aggregates will evolve slowly to form Ouzo droplets, and ultimately a separate phase. Investigation of water/THF/BODIPY: case of a solid fluorescent solute. Until now, most studies on the Ouzo effect have dealt with either oils or polymers which leave little doubt about the physical state of the dispersed phase (i.e. liquid for oils and solid for polymers). One noticeable exception is the case of the system water/ethanol/anethol (model system for the Ouzo or Pastis beverages), the melting point of anethol being around 21°C [START_REF] Grillo | Small-angle neutron scattering study of a world-wide known emulsion: Le Pastis[END_REF][START_REF] Sitnikova | Spontaneously Formed trans -Anethol/Water/Alcohol Emulsions: Mechanism of Formation and Stability[END_REF]. The BHT is another example of low molecular weight solid solute. In these cases, the physical state of the particles depends on their composition. Indeed, the phases of a separated ternary system can contain different amounts of the three constituents. It is generally assumed that in the case of Ouzo emulsions the droplets consist mostly of solute, but they may be more or less swollen with the solvent and even contain traces of water. The most rigorous method to determine the composition of the phases in equilibrium is to build the tie lines in the phase diagram [START_REF] Arce | Revising Concepts on Liquid-Liquid Extraction: Data Treatment and Data Reliability[END_REF]. However, this method is not applicable when one of the compounds is at extremely low concentration, as it is the case here. The above results concerning the NMR study of the water/THF/BHT system show that the dispersed phase in the Ouzo domain can remain liquid. Here, we took advantage of a fluorescent dye, that is emissive in the liquid and the solid states with different wavelengthes, to investigate the physical state of a solid solute through the phase diagram. We used a home made BODIPY dye (Figure 5) that yields a strong green emission when solubilized in organic solvents and a weaker red emission in the solid state (Figure SI-9) [START_REF] Goubault | The Ouzo effect: A tool to elaborate high-payload nanocapsules[END_REF]. We investigated the water/THF/BODIPY system. In contrast to previous systems, it was not possible to determine the cloudiness curve because of the fluorescence of BODIPY. Instead, we displayed the color of the samples under UV illumination in a "diagram of emission" (Figure 5). Very dilute samples ( BODIPY < 10 -6 ) look dark because they emit weakly. As expected, green emission is more intense in the solvent rich region (up-left part of the diagram). When increasing the amount of dye, the green emission decreases at the benefit of the red emission, indicating the presence of solid BODIPY particles. We used SLMS stability analysis (Figure SI-10) along the composition lines  THF =0.1, 0.2, 0.3 and 0.4 to build the binodal curve. Note that the SMLS uses a NIR laser at 880 m, which is far away from the excitation range of the BODIPY. For each line, we determined the boundary between stable and evolving samples. As previously, this draws a linear curve that we superimposed on the emission diagram in the Figure 5. Most samples along the binodal curve emit green light. We centrifugated two samples beyond the binodal, presenting yellow (f 1 :  THF =0.2;  BODIPY =2. 5 10 - 5 ) and red (f 2 :  THF =0.2;  BODIPY =2.5 10 -4 ) emission. Very interestingly, their pellets emit yellow and red light under irradiation for samples f 1 and f 2 respectively (Figure 5), indicating that the composition of the dispersed phase evolves from BODIPY swollen with solvent to pure solid BODIPY.  THF =0.2;  BODIPY =2. 5 10 -5 ; f 2 :  THF =0.2;  BODIPY =2. 5 10 -4 ).

NTA analysis proved to be limited to very dilute samples due to the excitation of BODIPY fluorescence at the operating wavelength (405 nm), but DLS measurements with laser wavelength of 638 nm could be achieved. Therefore, we monitored the samples for 3 hours after preparation using DLS. The results for the composition line  THF = 0.2 are displayed in Figure 6a. The first observation is that there exists a SFME domain in the range ( BODIPY = 10 -7 -2.5 10 -6 ), with stable droplets of diameter ca 100 nm, that remain stable in time. At larger concentrations (5.10 -6 and beyond), the size evolves with time. These observations are consistent with the SMLS measurements and confirm the location of the binodal curve. Finally, we measured the electrophoretic mobility of samples along the dilution line  THF =0.2 (Figure 6b) using an instrument operating at 633 nm. As previously, we find a maximum of (negative) mobility at the binodal, related to a strongly negative charge at the interface. As previously, the evolution of electrophoretic mobility is consistent with that the colloidal stability as observed with the DLS and SMLS analysis.

4.Conclusion

As an attempt to clarify the general behavior of Ouzo emulsions and the methodology to determine unambiguously the binodal curve, we achieved a careful examination of four ternary systems, with low molecular weight solutes, both liquid and solids. In contrast to many other studies, we did not make use of surfactant to avoid any interference with the pristine systems. We hypothesized that combining conventional DLS analysis with stability monitoring would allow us to delineate the Ouzo domain. This assumption is not obvious, as homogeneous supersaturated states can exist in a metastable zone. However, in the case of the Ouzo effect, nucleation is initiated since by definition particles are present. It is the growth of the droplets that is kinetically blocked or slowed down [START_REF] Sitnikova | Spontaneously Formed trans -Anethol/Water/Alcohol Emulsions: Mechanism of Formation and Stability[END_REF]. Thus, stability studies using SMLS, but also NTA or DLS, have allowed us to effectively discriminate Ouzo domain aggregates (more or less slowly evolving) from SFME aggregates (thermodynamically stable), validating this original approach. In most studies to date, phase diagrams have been constructed on the basis of visual observation of cloudiness and/or size analysis by scanning electron microscopy (SEM), DLS or other size analysis techniques [START_REF] Vitale | Liquid Droplet Dispersions Formed by Homogeneous Liquid-Liquid Nucleation: "The Ouzo Effect[END_REF][START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Beck-Broichsitter | Solvent selection causes remarkable shifts of the "Ouzo region" for poly(lactide-co-glycolide) nanoparticles prepared by nanoprecipitation[END_REF][START_REF] Chapel | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF]. In addition, surfactants can be added to the system to ensure colloidal stability of the particles during the analysis time [START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Marcus | Influence of additives on the structure of surfactant-free microemulsions[END_REF]. Consequently, the criteria for defining the Ouzo domain have been variable. Thus, the boundary between the monophasic and the Ouzo domains is often taken by construction as the onset of cloudiness, determined by visual observation. In other cases, the measurement of the mean hydrodynamic radius was used as a criterion [START_REF] Yan | Modular construction of singlecomponent polymer nanocapsules through a one-step surfactant-free microemulsion templated synthesis[END_REF][START_REF] Goubault | Effect of nanoparticles on spontaneous Ouzo emulsification[END_REF]. Indeed, the evolution of this diameter with the solute concentration shows two regimes: a constant regime and a regime where the size increases with the solute concentration. The transition between these two regimes has been interpreted as the boundary between SFME and Ouzo. Note that several works are based on the assumption that an Ouzo system should be cloudy and do not provide an analysis of the transparent zone [START_REF] Vitale | Liquid Droplet Dispersions Formed by Homogeneous Liquid-Liquid Nucleation: "The Ouzo Effect[END_REF][START_REF] Beck-Broichsitter | Preparation of nanoparticles by solvent displacement for drug delivery: A shift in the "ouzo region" upon drug loading[END_REF]. Regarding the high-concentration boundary of the Ouzo domain, SEM-based studies (mostly in the case of polymers) have used a sizedispersity criterion, to define the "stable" vs "unstable" Ouzo domains [START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF][START_REF] Chapel | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF]. In some other cases, compositions that were cloudy but not demixed after a "certain time" were considered as Ouzo emulsions [START_REF] Tehrani-Bagha | An Ouzo emulsion of toluene in water characterized by NMR diffusometry and static multiple light scattering[END_REF]. While we have addressed the case of low molecular weight solute (solid or liquid), many other studies have focused on nanoprecipitation of polymers by solvent-shifting. Significant differences can be noted. Indeed, in the case of polymers, the solubility limit is not very dependent on the polymer concentration, which would be reflected by an almost horizontal line in the representation of figure 1a [START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF][START_REF] Chapel | Impact of the Formulation Pathway on the Colloidal State and Crystallinity of Poly-ε-caprolactone Particles Prepared by Solvent Displacement[END_REF]. Moreover, detailed studies of the dependence of particle size with solvent and/or polymer concentration showed that polymer particles form by a nucleation-aggregation mechanism, favoring a narrow size distribution [START_REF] Aubry | Nanoprecipitation of Polymethylmethacrylate by Solvent Shifting:1[END_REF]. In fact, the surface area of the domain where particles with a low size distribution are observed is generally much larger than with low molecular weight compounds. It is our opinion that in the case of polymers, nucleation-growth readily yields particles of homogeneous size (which are termed "stable Ouzo"), and that polydisperse compositions containing micron-sized particles (termed "unstable Ouzo") appear in the vicinity of the spinodal. It should also be noted that surfactants are commonly used to limit the aggregation of polymer particles and can influence the nucleation conditions, in particular by reducing the critical radius of nucleation [START_REF] Lepeltier | Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices[END_REF][START_REF] Beck-Broichsitter | Solvent selection causes remarkable shifts of the "Ouzo region" for poly(lactide-co-glycolide) nanoparticles prepared by nanoprecipitation[END_REF]. The control of the physical state of nanoprecipitates (liquid, amorphous solid or crystalline) is very important, as it governs the solubility and therapeutic properties of certain drugs. Although most studies show that nanoprecipitation rarely leads to a crystalline structure, the reasons for this are still debated [START_REF] Lepeltier | Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices[END_REF]. One hypothesis is the presence of solvent, acting as a plasticizer of the particles, but experimental evidence is lacking. The SANS technique, particularly with contrast variation, could be used in combination with models to determine the composition and structure (homogeneous, core-shell) of the droplets/particles at moderate dilutions [START_REF] Prévost | Spontaneous Ouzo Emulsions Coexist with Pre-Ouzo Ultraflexible Microemulsions[END_REF][START_REF] Li | Large-Scale Structures in Tetrahydrofuran-Water Mixture with a Trace Amount of Antioxidant Butylhydroxytoluene (BHT)[END_REF]. Thus, one previous SANS study suggested that the aggregates formed in the SFME domain of the D 2 O/THF/BHT system consist of a small BHT core surrounded by a large THF shell [START_REF] Li | Large-Scale Structures in Tetrahydrofuran-Water Mixture with a Trace Amount of Antioxidant Butylhydroxytoluene (BHT)[END_REF]. Nevertheless, our NMR and fluorescence measurements here clearly established the presence of solvent with the solute in the dispersed phase. Note that the few previous NMR studies of the Ouzo effect, were carried out on liquid solutes at 25°C (anethole or toluene) and aimed to characterize the exchange dynamics between the dispersed and continuous phases [START_REF] Tehrani-Bagha | An Ouzo emulsion of toluene in water characterized by NMR diffusometry and static multiple light scattering[END_REF][START_REF] Carteau | The "Ouzo effect": Following the spontaneous emulsification of trans-anethole in water by NMR[END_REF]. The metastability of the droplets is one of the most intriguing features of the Ouzo emulsions. While droplet coalescence rate decreases with concentration, the generally high dilution of Ouzo domain is not enough to explain their stability, some times over months [START_REF] Sitnikova | Spontaneously Formed trans -Anethol/Water/Alcohol Emulsions: Mechanism of Formation and Stability[END_REF]. The adsorption of charged species at the interface has been proposed as an alternative explanation, and experimental evidences of the existence of a negative surface charge on emulsions produced by solvent-shifting were provided [START_REF] Ganachaud | Central Role of Bicarbonate Anions in Charging Water/Hydrophobic Interfaces[END_REF][START_REF] Roger | Why Are Hydrophobic/Water Interfaces Negatively Charged?[END_REF][START_REF] Ma | Impact of Spontaneously Adsorbed Hydroxide Ions on Emulsification via Solvent Shifting[END_REF]. This surface charge obviously has a pH signature and appears to be due either to the direct adsorption of endogenous CO 3 2-, HCO 3-or OH -ions at the interface or to the reaction of hydroxyl ions with acidic impurities. Here, for the first time, we follow the evolution of the charge with hydrophobic solute composition, from the SFME domain to the Ouzo domain, and show a regime change at the binodal, which can be related to a change in the composition of the dispersed objects.

In summary, the combination of conventional (DLS, Nanoparticle Tracking Analysis or NTA) and less conventional (NMR, stability analysis by SMLS, zeta potential, fluorescence) techniques allowed us to challenge conventional views on spontaneous emulsifications. We show that all four systems exhibit a SFME domain, separated from the Ouzo domain by the binodal curve. There is no discontinuity of size at the binodal but this transition can be determined by monitoring the colloidal stability of the sample. In addition, the droplets bear a spontaneous negative charge that shows a maximum at the binodal. Importantly, we clearly evidence that Ouzo emulsions can be transparent, and that the finest emulsions are generated in the transparent domain in between the binodal and the cloudiness curves. The cloudiness curve marks the abrupt increase in polydispersity. These results call into question the method of visual observation to identify the Ouzo domain, widely used in nanoprecipitation processes, and can be used as a rational guide for the design of nanoparticles and nanocapsules with polymers, drugs, or inorganic materials. There is still much to be understood about the transition between the SFME and Ouzo domains. We believe that future works should focus on analyzing both the composition and the structure of the dispersed phase in both domains. Short times kinetic experiments (using stopped-flow devices) may also provide interesting views on the early moments of droplet nucleation.

Figure 2 : 2 (c 2 :

 222 Figure 2: (A) 1D 1 H NMR spectra of BHT (a) solubilized in THF-d8 and (b-e) for samples along the composition line  THFd8 = 0.2 (c 2 : BHT = 10 -5 ; c 3 : BHT = 1.5 10 -5 ; c 4 : BHT = 3 10 -5 ; c 6 : BHT = 5 10 -5 ). The two sets of BHT proton resonances are identified by full and open symbols (▲/ , ♦/◊ and / ) as indicated on scheme. Solvent peaks are identified by filled stars: H 2 O ( ) and

Figure 3 :

 3 Figure 3: Electrophoretic mobility in function of the normalized concentration in BHT, for two compositions of THF (○:  THF = 0.1 and •:  THF = 0.2).  BHT * represents the BHT mass fraction at the binodal. Error bars represent the standard deviation of 3 measurements.

Figure 4 :

 4 Figure 4: Molecular formula of the solutes; mean hydrodynamical diameter (D H ) from NTA plotted in function of the solute mass fraction () normalized by the corresponding mass fractions at the cloudiness curve (**); and phase diagrams build through visual observation (light grey= limpid; dark grey = diphasic; hatched= cloudy).

Figure 5 :

 5 Figure 5: Chemical structure of the BODIPY dye and diagram of fluorescence emission ( exc =365 nm). The dashed line is the limit between stable and evolving samples as determined by SMLS. f 1 and f 2 represent the compositions that were centrifugated (f 1 :

Figure 6 :

 6 Figure 6: Investigation of the composition line  THF = 0.2 of the system water/THF/BODIPY (a) DLS measurements at initial time (──); after 1 h (---); after 3 h (‧‧‧‧‧‧). (b) Electrophoretic mobility of the dispersed phase in function of the mass fraction of BODIPY normalized by the fraction at the binodal.

Table 1 :

 1 Composition of the samples of series a

			0.15
		-5		-	-
	c 0	2.5 × 10 -6		85.3 ± 2.1	92.5 ± 5.9
	c 1	5 × 10 -6		87.5 ± 2.7	89.9 ± 8.4
	c 2	1 × 10 -5		93.5 ± 0.6	107.0 ± 2.1
	c 3 c 4	1.5 × 10 -5 2 × 10 -5	0.2	95.2 ± 4.7 69.9 ± 4.3	98.7 ± 1.5 156.6 ± 3.0
	c 5	3 × 10 -5		-	-
	c 6	5 × 10 -5		231.9 ± 13.8	404.6 ± 28.6
	c 7	1 × 10 -4		-	-

n -c n in the water/THF/BHT system, and mean hydrodynamic diameter of the distribution (D H ), as obtained by NTA at t=0 and t=3 h.

Acknowledgment:

We wish to thank the region Bretagne (ARED-107/ NAPOLI), the ANR (grant: 2020-CES06-OuzoFan), the CNRS, the University Rennes 1 and the platform ScanMat (2Cbiomif) for funding. Part of this work has been performed using the PRISM core facility (Biogenouest, Univ Rennes, Univ Angers, INRAE, CNRS, FRANCE): the authors thank Sandrine Pottier and Dr. Arnaud Bondon for their precious help and expertise. We very warmly thank Olivier Mongin for providing the BODIPY and Anthony Szymczyk and Bertrand Lefeuvre for complimentary access to the DLS.

Graphical abstract

Shedding Light on the Formation and Stability of Mesostructures in

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: