Smadar Gertel
email: smadar.gertel@sheba.health.gov.il

Gidi Karmon

Sivan Vainer

Ora Shovman

Martin Cornillet

Guy Serre

Yehuda Shoenfeld

Howard Amital

Immunomodulation of RA Patients' PBMC with a Multiepitope Peptide Derived from Citrullinated Autoantigens

Citrullinated peptides are used for measuring anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA). Accumulation of citrullinated proteins in the inflamed synovium suggests that they may be good targets for inducing peripheral tolerance. In view of the multiplicity of citrullinated autoantigens described as ACPA targets, we generated a multiepitope citrullinated peptide (Cit-ME) from the sequences of major citrullinated autoantigens: filaggrin, β-fibrinogen, vimentin, and collagen type II. We assessed the ability of Cit-ME or the citrullinated β60-74 fibrinogen peptide (β60-74-Fib-Cit) which bears immunodominant citrullinated epitopes (i) to modify cytokine gene expression and (ii) to modulate Treg and Th17 subsets in PBMC derived from newly diagnosed untreated RA patients. RA patient's PBMC incubated with Cit-ME or β60-74-Fib-Cit, showed upregulation of TGF-β expression (16% and 8%, resp.), and increased CD4 + Foxp3 + Treg (22% and 19%, resp.). Both peptides were shown to downregulate the TNF-α and IL-1β expression; in addition, Cit-ME reduced CD3 + IL17 + T cells. We showed that citrullinated peptides can modulate the expression of anti-and proinflammatory cytokines in PBMC from RA patients as well as the proportions of Treg and Th17 cells. These results indicate that citrullinated peptides could be active in vivo and therefore might be used as immunoregulatory agents in RA patients.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease estimated to affect nearly 1% of the adult population worldwide. It is characterized by progressive synovial inflammation resulting in irreversible joint destruction [START_REF] Imboden | The immunopathogenesis of rheumatoid arthritis[END_REF]. Many of the RA-specific autoantibodies are generated against citrullinated antigens and termed anticitrullinated protein antibodies (ACPA). These autoantibodies present a high diagnostic and prognostic value [START_REF] Nielen | Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors[END_REF][START_REF] Whiting | Systematic review: accuracy of anti-citrullinated peptide antibodies for diagnosing rheumatoid arthritis[END_REF][START_REF] Willemze | The ACPA recognition profile and subgrouping of ACPA-positive RA patients[END_REF]. The targets for ACPA are citrullinated peptides, that is, peptides that underwent posttranslation conversion of arginine to citrulline residues catalyzed by peptidylarginine deiminases (PAD). Currently, citrullinated peptides serve merely as immunosorbents in the ACPA diagnostic assays. The four citrullinated proteins, filaggrin, fibrinogen, vimentin, and collagen type II, have been described as the major autoantigens in RA [START_REF] Masson-Bessiere | The major synovial targets of the rheumatoid arthritisspecific antifilaggrin autoantibodies are deiminated forms of the alpha-and beta-chains of fibrin[END_REF][START_REF] Simon | The cytokeratin filament-aggregating protein filaggrin is the target of the so-called "antikeratin antibodies," autoantibodies specific for rheumatoid arthritis[END_REF][START_REF] Vossenaar | Rheumatoid arthritis specific anti-Sa antibodies target citrullinated vimentin[END_REF].

The pathogenesis of RA appears to be based on inflammatory responses mediated by self-reactive T and B cells reacting against citrullinated proteins. In autoimmune conditions, T cells reactive to self-antigens escape elimination in the thymus and are activated in the periphery where they can damage specific organs.

PAD isoforms are expressed in thymic epithelial cells; however, it is not clear whether a negative selection of citrulline-specific T cells occurs and whether those T cell clones manage to escape into the periphery [START_REF] Engelmann | The prerequisites for central tolerance induction against citrullinated proteins in the mouse[END_REF]. In addition, the presence of ACPA is associated with RA susceptibility alleles such as HLA-DRB1, HLA-DRB4, and HLA-DRB10 genes [START_REF] Auger | Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen[END_REF][START_REF] Gonzalez-Gay | Influence of human leukocyte antigen-DRB1 on the susceptibility and severity of rheumatoid arthritis[END_REF]. These alleles encode for a specific motif in the peptide-binding pocket of the human leucocyte antigen (HLA) bearing the so-called shared epitope (SE) [START_REF] Gregersen | The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis[END_REF]. Citrullination may enhance the immunogenicity of "altered-self" peptides through increased binding affinity to SE-containing HLA-DRB presented on MHC class II molecules. This may result in loss of tolerance to the related citrullinated antigens, mediated by activation of citrullinespecific-autoreactive T and B cells, leading to production of ACPA, then formation of immune complexes in the synovial tissue, and induction of the proinflammatory cytokine secretion by macrophages, all leading to synovial inflammation [START_REF] Auger | Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen[END_REF][START_REF] Hill | Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule[END_REF][START_REF] Law | T-cell autoreactivity to citrullinated autoantigenic peptides in rheumatoid arthritis patients carrying HLA-DRB1 shared epitope alleles[END_REF][START_REF] Van Der Helm-Van Mil | The HLA-DRB1 shared epitope alleles are primarily a risk factor for anti-cyclic citrullinated peptide antibodies and are not an independent risk factor for development of rheumatoid arthritis[END_REF].

The inflamed joints in RA contain a large number of T cells, the majority of which are CD4 + T cells [START_REF] Striebich | Selective accumulation of related CD4+ T cell clones in the synovial fluid of patients with rheumatoid arthritis[END_REF]. The autoantigen that causes stimulation and expansion of specific T cell subsets should be present in the joint and presented by the HLA-DR alleles associated with RA. The fact that inflamed joints of RA patients contain large amounts of citrullinated proteins implies that they may be possible targets for tolerance induction [START_REF] Turunen | Different amounts of proteinbound citrulline and homocitrulline in foot joint tissues of a patient with anti-citrullinated protein antibody positive erosive rheumatoid arthritis[END_REF]. Restoring self-tolerance without causing immunosuppression remains a major challenge.

In view of the multiplicity of citrullinated proteins described as target autoantigens for ACPA, we generated a multiepitope citrullinated peptide (Cit-ME) derived from the sequence of the major prevalent autoantigenic citrullinated proteins, namely, filaggrin, β-fibrinogen, vimentin, and collagen type II. Previously, we reported that the Cit-ME peptide ameliorates adjuvant-induced arthritis (AIA) in rats via induction of regulatory T cells and downregulation of Th17 cells [START_REF] Gertel | Immune tolerance induction with multiepitope peptide derived from citrullinated autoantigens attenuates arthritis manifestations in adjuvant arthritis rats[END_REF][START_REF] Gertel | Tolerogenic citrullinated peptide for arthritis[END_REF].

The current research analyzed the capacity of the same citrullinated multiepitope peptide, Cit-ME and of an additional single citrullinated fibrinogen peptide bearing immunodominant epitopes, to modify cytokine expression and modulate T cell subsets in PBMC from patients with RA.

Materials and Methods

Human Subjects and Sample Procurement.

A total of 30 patients with RA (17 females and 13 males) were included in the study. All patients fulfilled the 2010 American College of Rheumatology (ACR) criteria for RA. The study protocol was approved by the Medical Ethics Review Board of Sheba Medical Center. All participants signed an informed consent form prior to the initiation of the study. We enrolled only newly diagnosed patients. Patients' age was 52.9 ± 13.7 (mean ± standard deviation) years, and 90% were ACPA positive (Table 1). Blood samples were collected before starting any therapy. The study was designed to test immunomodulation induced by citrullinated peptides and to assess their possible contribution to the pathogenesis of arthritis. The suggested controls were approved biological disease modifying antirheumatic drugs; therefore, the assays were conducted only on PBMC derived from patients with RA.

Detection of Antictrullinated Protein Antibodies (ACPA).

Sera from patients were tested for ACPA using the commercial ELISA QUANTA Lite® CCP3 IgG kit (Inova, USA), according to the manufacturer's instructions. Sera with results < 25 U/mL are defined as negative, and sera with results ≥ 25 U/mL, as positive.

Synthetic Peptides and Biotherapeutic

Agents. The multiepitope citrullinated peptide Cit-ME (Figure 1) and its noncitrullinated counterpart containing arginine instead of citrulline residues, Non-Cit-ME, were obtained from GL Biochem (Shanghai, China). The citrullinated peptide β-Fib-Cit (β60-74Cit 60, 72, 74 ) with the sequence CitPAPPPISGGGYCi-tACit derived from the β chain of human fibrinogen [START_REF] Iobagiu | The antigen specificity of the rheumatoid arthritis-associated ACPA directed to citrullinated fibrin is very closely restricted[END_REF] and its noncitrullinated counterpart (β-Fib-NC) were obtained from NeoMPS (Strasbourg, France). The sequences of β-Fib-Cit and β-Fib-NC are also present in part in Cit-ME and Non-Cit-ME, respectively.

All peptides were added to cell culture medium at a concentration of 1.25 μg/mL. This concentration was selected based on previous publications dealing with the capability of autoantigen peptides to induce proliferation at various concentrations of 2.5-10 ug/mL [START_REF] Aggarwal | T cell responses to citrullinated self-peptides in patients with rheumatoid arthritis[END_REF][START_REF] Tandon | T cell responses to synthetic TSH receptor peptides in Graves' disease[END_REF]. We determined no proliferation at a concentration of 1.25 μg/mL with different citrullinated peptides (data not shown); hence, this concentration was selected to confirm tolerizing regime. In the same way, infliximab or tocilizumab was added to cell culture medium at a concentration of 10 μg/mL.

In Vitro Experiments.

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood using lymphoprep (Amersham). Cells were cultured in RPMI 1640 medium containing 10% bovine fetal serum supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), 2 mmol/L L-glutamine, and 50 μM 2-β-mercaptoethanol. Cells were incubated for 24-72 hours at 37 °C with the experimental peptides, drugs, or medium alone as control.

Tregs and Th17

Staining. Tregs were identified by the analysis of CD4 and Foxp3 expression. Cells were labeled with anti-CD4 (eBioscience Inc., San Diego, CA, USA), then fixed and permeabilized with Foxp3 detection kit buffers, and stained with anti-Foxp3. In some experiments, anti-CD25 was used to identify CD4 + CD25 + Foxp3 + cells. Th17 cells were determined by addition of phorbol 12-myristate 13acetate (PMA) (5 ng/mL) and ionomycin (1 μM) (Sigma-Aldrich, Seelze, Germany) to the culture media for 6 hours. Monensin (eBioscience Inc.) was added in the last two hours of stimulation. For Th17 analysis, cells were stained with anti-CD3, followed by fixation and permeabilization (eBioscience Inc.), and then stained intracellularly with anti-IL-17. Cells were analyzed by a flow cytometer (FACScalibur, Becton Dickinson, Franklin Lakes, NJ, USA). Data analysis was conducted using FlowJo software (Tree Star, Ashland, OR, USA).

2.6. Real-Time PCR. RNA was isolated using the Total RNA Purification Plus Kit (Norgen Biotek, ON, CA) according to the manufacturer's instructions. For cDNA synthesis, 1 μg total RNA was transcribed with cDNA transcription reagents using the High Capacity cDNA Reverse Transcription Kit (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer's instructions. Gene expression was measured in real-time PCR performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Primer sequences (forward and reverse, resp.) were the following: human TGF-β 5 ′ -GACACCAACTATTGCTTCAG-3 ′ and

Results

Low Doses of Citrullinated Peptides Change Gene Expression of Anti-and Proinflammatory Cytokines in

Cultured PBMC from RA Patients. To detect whether the multiepitope citrullinated peptide Cit-ME and the individual citrullinated peptide β-Fib-Cit could induce tolerance through immunomodulation of immune cells in RA, we studied their in vitro effects on cytokine gene expression and T cell subsets of cultured PBMC obtained from RA patients. The PBMC were cultured for 24 hours in the absence or presence of the citrullinated peptides and as controls their arginine counterparts, at a concentration of 1.25 μg/mL or with infliximab at 10 μg/mL. Thereafter, mRNA was isolated from the cells, and the level of TGF-β, TNF-α, and IL-1β gene expression was determined by real-time RT-PCR.

As shown in Figure 2(a), Cit-ME peptide significantly upregulated the TGF-β gene expression level in the PBMC (p = 0 04) compared to incubation of cells with medium alone (normalization was performed to GAPDH and medium control was considered as 1). The peptide β-Fib-Cit seemed to also upregulate TGF-β gene expression, but this was not significant with the small number of patients studied. None of the noncitrullinated peptides produced any upregulation of the TGF-β gene expression compared to the medium. In contrast, incubation of cells with infliximab resulted in decreased TGF-β expression, compared to medium control.

Concomitantly, the Cit-ME peptide significantly downregulated the proinflammatory TNF-α and IL-1β expression levels as compared to the medium control (p = 0 03 and p = 0 05, resp.), as shown in Figure 2 TNF-α level but reduced the mean IL-1β level compared to the medium control (p = 0 07). medium alone, but as compared to β-Fib-NC (n = 6, average of 1.1 ± 0.04), variation was nonsignificant. Neither infliximab (n = 10, average of 1.07 ± 0.05) nor tocilizumab (n = 11, average of 0.99 ± 0.05) significantly altered the fold change of % CD4 + Foxp3 + T cells compared to the medium control. Higher expression of Foxp3 on gated CD4 T cells in PBMC of representative RA patient that were incubated with Cit-ME compared to medium alone is shown (Figure 4(c)). This was true also for CD4 + CD25 + Foxp3 + cells that were elevated in RA patients' PBMC incubated with Cit-ME (8.3%) compared to Non-Cit-ME or medium control (7.4%) (Figure 4(d)). Figure 5(a) shows representative plot of ACPA + -RA patient PBMC stained with anti-CD3 and anti-IL-17. As shown in Figure 5(b), after incubation with the Cit-ME peptide, significant reduction in the fold change of % CD3 + IL-17 + T cells was detected (n = 9, average of 0.77 ± 0.08) compared to the medium control (n = 9, considered as 1) (p = 0 05). Incubation with the Non-Cit-ME peptide resulted in reduction in the fold change of % CD3 + IL-17 + T cells (n = 7, average of 0.9 ± 0.1) as compared to the medium control alone, but the variations were nonsignificant.

The

Gene expression ratio

훽-Fib-Cit 훽-Fib-NC Cit-ME Non-Cit-ME Infliximab Medium 훽-Fib-Cit 훽-Fib-NC Cit-ME Non-Cit-ME Infliximab (b)

Discussion

We have shown that Cit-ME, a multiepitope citrullinated peptide, has the capacity to immunomodulate pro-and anti-inflammatory gene expression in RA patients' PBMC. In vitro Cit-ME induced Treg and decreased the number of Th17 cells. In a previous study, we demonstrated that the tolerogenic Cit-ME peptide ameliorated manifestations of arthritis in a rat model of adjuvant-induced arthritis (AIA). The mechanisms leading to suppression of arthritis by Cit-ME peptide in vivo were also mediated by altering the Treg/Th17 balance and by increasing T cell apoptosis [START_REF] Gertel | Immune tolerance induction with multiepitope peptide derived from citrullinated autoantigens attenuates arthritis manifestations in adjuvant arthritis rats[END_REF].

ACPA are known to appear years prior to arthritis, and citrullinated proteins are assumed to be the original target antigens in RA and perhaps the immunogens [START_REF] Sebbag | Clinical and pathophysiological significance of the autoimmune response to citrullinated proteins in rheumatoid arthritis[END_REF]. Hence, they might be the key to restoring immune tolerance and prevent impending disease. The approach we present here is based on inducing peripheral tolerance using a synthetic peptide derived from sequences of multiple prevalent citrullinated autoantigens. Indeed, the citrullinated β-fibrinogen peptide was also able to immunomodulate immune cells of RA patients even if to a limited extent compared to the Cit-ME peptide.

Stimulation of PBMC from 51 RA patients with the 5 different citrullinated fibrinogen peptides (50 μg/mL) demonstrated that they significantly suppressed the T cell proliferative responses and did not change the production levels of IFN-γ and IL-17 [START_REF] Shin | Role of citrullinated fibrinogen peptides in the activation of CD4 T cells from patients with rheumatoid arthritis[END_REF]. These findings clearly indicated that the citrullinated fibrinogen peptides did not induce an immunogenic response but rather generated a tolerogenic phenotype.

Moreover, in a clinical trial conducted on ACPA + -RA patients carrying HLA-DRB1 SE allele's, immunotherapy of autologous dendritic cells loaded with four different citrullinated peptide antigens improved a patient's clinical state. The observed effects were associated with reduction of effector T cells and reduced proinflammatory cytokines in the serum with an increased ratio of regulatory to effector T cells [START_REF] Benham | Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype-positive rheumatoid arthritis patients[END_REF].

On the other hand, studies that evaluated immune responses towards different citrullinated proteins, such as citrullinated vimentin and citrullinated aggrecan in RA patients, demonstrated a certain degree of immune cell stimulation directed to these citrullinated proteins [START_REF] Feitsma | Identification of citrullinated vimentin peptides as T cell epitopes in HLA-DR4-positive patients with rheumatoid arthritis[END_REF][START_REF] Delwig | Response of Th17 cells to a citrullinated arthritogenic aggrecan peptide in patients with rheumatoid arthritis[END_REF].

The dose of autoantigen used is a critical parameter, vimentin and aggrecan citrullinated peptides could induce certain proliferative response in T cells from RA patients at a concentation range of 2.5-10 μg/mL [START_REF] Aggarwal | T cell responses to citrullinated self-peptides in patients with rheumatoid arthritis[END_REF]. Also, 5 μg/mL of TSH peptides induce T cell response in vitro in PBMC derived from Graves' disease patients [START_REF] Tandon | T cell responses to synthetic TSH receptor peptides in Graves' disease[END_REF]. Here, citrullinated peptides were administered at a concentration of 1.25 μg/mL chosen on a basis of their potential ability of tolerance induction. Possibly, such an approach induces TCR signals that are insufficient to fully activate T cells and, therefore, lead to functional T cell suppression by either energy or clonal deletion [START_REF] Turley | Prospects for antigen-specific tolerance based therapies for the treatment of multiple sclerosis[END_REF].

It was shown that RA patients have significantly higher frequencies of Cit-specific T cells and that a greater proportion of these cells displays a Th1 memory phenotype [START_REF] James | Citrulline-specific Th1 cells are increased in rheumatoid arthritis and their frequency is influenced by disease duration and therapy[END_REF]. Thus, the administration of low-dose citrullinated peptides might assist to reverse the pathogenic autoreactive immune response by inducing immunomodulation. We previously reported that Cit-ME effectively inhibited the course of AIA in Lewis rats. These therapeutic effects were associated with increased Treg and T cell apoptosis and fewer Th17 cells in the treated rats [START_REF] Gertel | Immune tolerance induction with multiepitope peptide derived from citrullinated autoantigens attenuates arthritis manifestations in adjuvant arthritis rats[END_REF].

Here, the in vitro effect of Cit-ME on CD4 + Foxp3 + and CD3 + IL-17 + T cells was assessed; Cit-ME and β-Fib-Cit upregulated the CD4 + Foxp3 + T cells. Further, characterizing the suppressive capability of such population is required. Phenotypical analysis of Treg into subgroups of activated Treg (CD45RA -Foxp3 high ), resting Treg (CD45RA + Fox-p3low), which are both suppressive in vitro, and cytokinesecreting nonsuppressive non-Treg (CD45RA -FoxP3 low ) as shown could be more informative [START_REF] Miyara | Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor[END_REF].

For instance, heat shock protein 60, altered peptide ligand (APL1), an additional autoantigen possibly involved in the pathogenesis of RA, was used for the induction of peripheral tolerance. Stimulation of PBMC from RA patients by APL1 increased the proportions of Treg [START_REF] Mdel | An altered peptide ligand corresponding to a novel epitope from heatshock protein 60 induces regulatory T cells and suppresses pathogenic response in an animal model of adjuvant-induced arthritis[END_REF], induced apoptosis of activated CD4 + T cells, presumably through a Tregdependent mechanism [START_REF] Barbera | APL-1, an altered peptide ligand derived from human heat-shock protein 60, selectively induces apoptosis in activated CD4+ CD25 + T cells from peripheral blood of rheumatoid arthritis patients[END_REF], and increased Treg suppression against APL1 responding effector T cells, at the same time decreased IL-17 that is produced by activated T cells [START_REF] Barbera | APL1, an altered peptide ligand derived from human heat-shock protein 60, increases the frequency of Tregs and its suppressive capacity against antigen responding effector CD4 + T cells from rheumatoid arthritis patients[END_REF].

Similarly, in systemic lupus erythematosus (SLE), abnormal apoptosis may lead to extracellular release of nucleosomes, which include various histone proteins that are recognized as neoepitopes [START_REF] Chabre | Presence of nucleosome-restricted antibodies in patients with systemic lupus erythematosus[END_REF][START_REF] Lu | Major peptide autoepitopes for nucleosome-specific T cells of human lupus[END_REF]. Supplementing PBMC cultures of lupus patients with histone nucleosomal peptides induced an increase of Treg and suppressed IFNα gene expression. Each of the histone peptidic epitopes also inhibited the pathogenic IgG autoantibody production in the lupus patients' PBMC. Combinations of these epitopes were even more efficient in suppressing pathogenic autoantibody production [START_REF] Zhang | Major pathogenic steps in human lupus can be effectively suppressed by nucleosomal histone peptide epitope-induced regulatory immunity[END_REF]. Accordingly, administration of low-dose histone-nucleosomal peptides induced tolerance in humans [START_REF] Kang | Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets[END_REF].

In our study, the anti-inflammatory response phenomenon exerted by the citrullinated peptides was compared to approved biological medications in RA (infliximab and tocilizumab). Addition of the anti-TNF-α agent infliximab did not affect significantly TGF-β, TNF-α, IL-8, and CCL-3 gene expression, while IL-1β mRNA was significantly decreased and IL-6 mRNA was elevated compared to the medium control. The observed effects on lymphocytes following stimulation with infliximab can indicate on the potential drug induce target genes in naïve RA patients' PBMC. We could not detect changes in Treg population following the addition of an anti-TNF-α (infliximab) or an anti-IL6 receptor agent (tocilizumab). However, anti-TNF therapy in RA patients was shown to have the capacity to generate newly differentiated population of Treg cells [START_REF] Nadkarni | Anti-TNFalpha therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-beta[END_REF].

Study limitations are the following: first, the immunomodulatory effects of the citrullinated peptides were compared to the noncitrullinated matched peptides. Although the latter peptides could not promote equivalent immunomodulation, we cannot exclude that such control peptides may have a biological effect since they possess sequence homologies to known autoantigens and might evoke certain immune responses. Scrambled peptides should be considered as a better control for Cit-ME and β60-74-Fib-Cit activity.

Second, the citrullinated peptides administered in our study could immunomodulate cytokine expression also in ACPA seronegative patients (n = 3), indicating that although the ACPA titers measured were below the cut-off of the commercial ELISA kit for ACPA detection, the citrullinated peptides could nevertheless stimulate the PBMC and alter the mRNA profile of the cytokines. Further studies are required to determine the mechanistic effects of citrullinated peptides in ACPA seronegative RA patients.

Conclusion

In the present study, we have shown that a multiepitope citrullinated peptide could be an attractive target for therapeutic intervention in RA patients. This peptide may stimulate the expression of anti-inflammatory cytokines and downregulate proinflammatory cytokines in T cells. These modifications may shift the Treg/Th17 imbalance and restore homeostasis in arthritis.

5 ′

 5 -CAGGCTCCAAATGTAGGG-3 ′ , TNF-α 5 ′ -CCCAGG GACCTCTCTCTAATCA-3 ′ and 5 ′ -GGTTTGCTACAACA TGGGCTACA-3 ′ , IL-1β 5 ′ -TGATGGCTTATTACAGTGG CAATG-3 ′ and 5 ′ -GTAGTGGTGGTGGGAGATTCG-3 ′ , IL -6 5 ′ -CAATCTGGATTCAATGAGGAGAC-3 ′ and 5 ′ -TGT TCCTCACTACTCAAATCT-3′, IL-8 5′-TGGCAGCCTTC CTGATTTCT-3′ and 5′-GGGTGGAAAGGTTTGGAGTA TG-3′ and CCL-3 5′-CCGGTGTCATCTTCCTAACC-3′ and 5′-TTCTGGACCCACTCCTCACT-3′, and GAPDH 5′-GA AGGTGAAGGTCGGAGTC-3 ′ and 5 ′ -GAAGATGGTGAT GGGATTTC-3 ′ . The expression level of a gene in a given sample was represented as 2

Figure 1 :

 1 Figure 1: Scheme of Cit-ME peptide. Representative scheme of the multiepitope citrullinated peptide sequence delineated from the major autoantigens of anticitrullinated protein antibody (ACPA). The citrullinated residues are indicated (Cit).

Figure 4 (

 4 a) shows representative plot of ACPA + -RA patient PBMC stained with anti-CD4 and anti-Foxp3. As shown in Figure4(b), Cit-ME significantly increased the fold change of % CD4 + Foxp3 + T cells (n = 13, average of 1.22 ± 0.06) compared to the Non-Cit-ME (n = 13, average of 1.0 ± 0.07) or medium (n = 12, considered as 1) (p = 0 02), respectively. In addition, incubation with the β-Fib-Cit peptide resulted in significant increase in fold change of % CD4 + Foxp3 + T cells (n = 6, average of 1.19 ± 0.07) as compared to

Figure 2 :

 2 Figure 2: Cit-ME peptide upregulates TGF-β and downregulates TNF-α and IL-1β mRNA expression. PBMC of RA patients (n = 12) were cultured (5 × 10 5 cells/well) for 24 hours in the presence of citrullinated peptides (1.25 μg/mL). TGF-β, TNF-α, and IL-1β mRNA gene expression was determined by real-time RT-PCR. Results are presented as the mean ± SE percentage of gene expression.

Figure 4 :

 4 Figure 4: Continued.

Figure 4 :

 4 Figure 4: Cit-ME upregulates Treg in vitro. (a) Representative dot plot of CD4 + Foxp3 + T cells from a RA patient. Positive staining is presented in the right upper quadrant of each graph with the percentage indicated. (b) The fold change of % CD4 + Foxp3 + T cells determined by flow cytometry (n = 6-13). Data are presented as mean values ( * p = 0 04, * * p = 0 02). (c) Histogram shows representative Foxp3 staining for one RA patient, gated for CD4 + T cells. (d) Representative dot plot of RA patient PBMC gated for CD4 and analyzed for CD25 and Foxp3 expression.

Figure 5 :

 5 Figure 5: Cit-ME downregulated Th17 cells in vitro. (a) Representative plots of CD3 and IL-17 staining. Positive staining is presented in the right upper quadrant of each plot with the percentage indicated. (b) Fold change of % CD3 + IL-17 + T cells (n = 11). Data are presented as mean values ( * p = 0 05).

Table 1 :

 1 Characterization of rheumatoid arthritis patients analyzed in the in vitro assays.

	Patient number Gender Age	ACPA status	ACPA value
	1	Female 35	Negative	12
	2	Female 58	Moderate	49
	3	Female 36	Moderate	48
	4	Female 32 Weakly positive	26
	5	Male	50	Highly positive	138
	6	Female 38	Positive	78
	7	Female 51	Negative	4
	8	Male	49	Highly positive	242
	9	Female 50	Positive	55
	10	Female 50	Highly positive	258
	11	Male	35	Highly positive	195
	12	Female 68	Positive	61
	13	Female 33	Highly positive	128
	14	Male	53	Highly positive	211
	15	Female 64	Highly positive	600
	16	Male	60	Negative	3
	17	Female 45	Positive	41
	18	Male	70	Highly positive	210
	19	Male	67	Highly positive	3700
	20	Male	57	Highly positive	191
	21	Female 55	Highly positive	171
	22	Female 48	Moderate	57
	23	Male	22	Highly positive	121
	24	Female 65	Highly positive	227
	25	Male	58	Negative	6
	26	Female 75	Highly positive	210
	27	Male	72	Highly positive	210
	28	Male	63	Highly positive	210
	29	Male	60	Highly positive	210
	30	Female 70	Highly positive	150

  -ΔΔCt where ΔΔCT = ΔCT experimental -ΔCT medium and ΔCT = CT experimental -CT housekeeping . The GAPDH levels were used to normalize gene expression levels.

2.7. Statistical Analysis. Statistical comparisons were conducted using the two-tailed Mann Whitney U test and Student t-test (GraphPad/Prism version 5 software, San Diego, CA, USA).

  Cit-ME Peptide Downregulates IL-6, IL-8, and CCL3 mRNA Expression in Cultured PBMC from RA Patients. Since Cit-ME peptide immunomodulates the expression of essential inflammatory genes, it was of interest to find out whether it might alter the expression of additional related genes.

		3.3. The Cit-ME Peptide Affects Treg and Th17 Proportions in
		Cultured PBMC from RA Patients. The effects of Cit-ME pep-
		tide on Treg were analyzed in cultured PBMC from RA patients. PBMC (2 × 10 6 ) were cultured with Cit-ME, β-Fib-
	We mea-	Cit, matched noncitrullinated peptides (Non-Cit-ME and
	sured cytokine gene expression typically involved in innate	β-Fib-NC), or medium alone. We also tested whether inflix-
	immune responses, namely, IL-6, IL-8, and CCL-3. Figure 3,	imab and tocilizumab could affect Treg proportion as well.
	represents cytokine gene expression levels (normalization was performed to GAPDH and medium control was consid-	Samples were analyzed by flow cytometry 72 hours after incubation, to measure the frequency of CD4 + Foxp3 + T cells.
	ered as 1). In accordance with the expression pattern of the
	proinflammatory TNF-α and IL-1β, incubation of Cit-ME

with PBMC of RA patients resulted in a significant downregulation in IL-6, IL-8, and CCL-3 gene expression as compared to incubation with medium alone (p < 0 02). However, the control peptide Non-Cit-ME could also downregulate significantly the IL-6 expression (p < 0 02) as compared to medium control. Whereas, the control peptide Non-Cit-ME or infiliximab could not downregulate significantly the IL-8 and CCL-3 expression as compared to medium control in cultured RA patients' PBMC.

  Figure 3: Cit-ME peptide downregulates IL-6, IL-8, and CCL-3 mRNA expression. PBMC of RA patients (n = 7-10) were cultured (5 × 10 5 cells/well) for 24 hours in the presence of Cit-ME (1.25 μg/mL). mRNA gene expression was determined by real-time RT-PCR. Results are presented as the mean ± SE percentage of gene expression, * p < 0 02.

	Gene expression ratio	0.4 0.8 4 6 8 CD4+	52.05 Cit-ME 52.45 37.67	Infliximab 훽-Fib-Cit IL-6 ⁎ 5.7% Non-Cit-ME 훽-Fib-NC 5.9%	5.66 54.87 Medium 1.01 53.66 5.88 37.67	Gene ratio expression	0.4 0.8 1.2	Cit-ME 6.7% Cit-ME Non-Cit-ME 5.3%	IL-8 6.70 51.88 ⁎ Infliximab Non-Cit-ME 5.30 57.62 0.77 43.04	Tocilizumab 4.3% ratio expression Gene Medium Infliximab 5.2%	4.31 1.2 0.8 0.4 5.22 0.77	Cit-ME 53.66	CCL-3 Medium Infliximab 5.0% Non-Cit-ME ⁎	Medium 5.02
			40.74		0.92	40.14				0.91	36.60		0.55	40.62	0.70
										Foxp3+

Acknowledgments

The authors thank the patients who participated in this study, and they want to acknowledge Dr. Boris Gilburd for preforming the ELISA using QUANTA Lite CCP3 (INOVA Diagnostics Inc.).

Conflicts of Interest

The authors have no financial conflicts of interest.


Venancio Bruno Robert

Calero Vila

Miguel-Angel Angel Suarez

Roland Salut

Florent Behague

Fadi Baida

Nadège Courjal

Maria-Pilar Bernal-Artajona

Venancio Calero

Nadege Courjal

Maria-Pilar Bernal
email: mp.bernal@femto-st.fr

Cost-efficient and high precision method for the assembly of LN-based photonic crystal slabs on the fiber tip for the implementation of E-field sensors

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

Introduction

Optical fibers are experiencing an important growth, in addition to data transport, also in the sensing field due to their unique versatility. They are nowadays employed in applications such as non-invasive medical surgery and structures damage monitoring [START_REF] Peterson | Fiber-optic sensors for biomedical applications[END_REF][START_REF] Zhou | Casing pipe damage detection with optical fiber sensors[END_REF]. Moreover, the measurement of other magnitudes such as temperature, refractive index, displacement, pressure and acceleration have been also demonstrated through the so-called Lab-on-Fiber technologies [START_REF] Chikh-Bled | Bahavior of femtosecond laser-induced eccentric fiber Bragg gratings at very high temperatures[END_REF]. Recently, the progress in micro-and nanofabrication techniques is making possible the growth of fiber tip sensors whose functional area consist on photonic nanostructures integrated on the fiber extremity such as photonic circuits [START_REF] Taillaert | Grating couplers as interface optical fibres and nanophotonic waveguides[END_REF], allowing a new era the of ultra-compact, versatile sensors. Moreover, Guided Resonances (GRs) have been recently implemented on the fiber tip [START_REF] Nottbohm | Mechanically stacked 1-nm-thick carbon nanosheets, ultrathin layered materials with tunable optical, chemical and electrical properties[END_REF][START_REF] Passoni | Grating couplers in silicon-on-insulator: the role of photonic guided resonances on lineshape and bandwidth[END_REF] due to the coupling efficiency of the fiber propagating mode with the photonic crystal (PhC) mode, in addition to the light localization and vertical confinement performances allowing light-matter interaction enhancement [START_REF] Brongersma | Light management for photovoltaics using high-index nanostructures[END_REF][START_REF] Piper | Total absorption in a graphene monolayer in the optical regime by critical coupling with a photonic crystal guided resonance[END_REF]. In a recent work from our group [START_REF] Calero | An ultra-side bandwidth, ultra-high spatial resolution and ultra compact electric field sensor based on Lab-on-fiber technology[END_REF], a GRbased fiber tip configuration is combined with lithium niobate (LN) in order to enhance its electro-optical performances producing an ultra-compact and distortion-free electric field sensing probe. Thus, a sub-micrometer free standing slab with a photonic crystal (PhC) is designed in a 700 nm TFLN membrane so that it can be integrated on the fiber tip. Despite the fact that the integration of bulk ferroelectric crystals on a fiber tip is a well-known approach [START_REF] Gaborit | A nonperturbative electrooptic sensor for in situ electric discharge characterization[END_REF] producing all-dielectric E-field sensing probes, the fiber tip approach based on the GRs has footprints that are thousands of times smaller than these massive crystal devices. The interest of reduced dimensions is very high since that, in addition to a submillimeter footprint, it offers clear advantages in terms of enhanced linearity with respect to the E-field, micrometer spatial resolution as well as extended bandwidth, only limited by the LN intrinsic bandwidth which is in the order of tens of THz. However, the nanofabrication of LN-based PhC with nanometric precision on LN is still limited to the use of the Focused Ion Beam (FIB) technology [START_REF] Calero | Towards highly reliable, precise and reproducible fabrication of photonic crystal slabs on lithium niobate[END_REF] or e-beam assisted IBEE techniques [START_REF] Geiss | Photonic microstructures in lithium niobate by potassium hydroxide assisted ion beam-enhanced etching[END_REF], which supposes a constraint on the PhC dimensions. Consequently, the mentioned fiber tip E-field sensor [START_REF] Calero | An ultra-side bandwidth, ultra-high spatial resolution and ultra compact electric field sensor based on Lab-on-fiber technology[END_REF] utilizes a PhC with an area of 19 μm × 19 μm which requires, due to its compact size, more complex and expensive systems such as the FIB system for the precise welding between the PCS and the fiber facet. This process, due to its serial nature, generally lasts several hours for each sensor head, leading to a low throughput-integration process remaining still far from massive production procedures. As an alternative, transfer-printing techniques based on epoxy solutions have been employed for reducing assembly costs for the welding [START_REF] Kim | Microstructures eleastomeric surfaces with reversible adhesion and examples of their use in deterministic aseembly by transfer printing[END_REF]. However, some constraints remain concerning the parallelism between the fiber and the photonic structure. A promising technique using a camera as feedback loop for the optimization of the angle between both surfaces has been recently reported [START_REF] Kudryavtsev | Analysis of cad model-based visual tracking for microassembly using a new block set for matlab/simulink[END_REF]. Nonetheless, this technique does not take any optical response information as feedback and, as a consequence, the device can only be tested at the end of the assembly. In this paper, we demonstrate a cost-efficient assembly method which simplifies the previously mentioned techniques. Thus, a system based on a stretchable sample holder ensures the parallelism between both surfaces when applying a vertical force, removing the need of complex rotational systems. In addition, an active positioning system is also implemented which allows, by observing the spectrum of the GRs during the assembly, the control of different parameters such as the beam centering. This novel assembly method can be easily used for the batch production of different GR-based fiber tip sensors, for a large range of applications, beyond E-field sensors, like the measurement of physical parameters such as pressure, refractive index, temperature, etc.

The stretchable membrane concept

The fiber-tip E-field sensor developed in this work is sketched in Fig. 1(a,b). An optical fiber is pigtailed by a ferrule to obtain a surface wide enough to act as a support for 3mm-diameter photonic chip which represents the sensing part of the device. The chip contains a PhC structure which is designed on a 700nm-thick membrane of X-cut LN centered to the optical fiber core. The membrane is based on a rigid 380μm-thick substrate where only a 250μm-diameter circle located at its center defines the shape of the free-standing membrane. This configuration acts as a protection crown for the PhC whose sensor performances depend on. The PhC geometry study and optimization are beyond the scope of this work but explained in detail in [START_REF] Qiu | Guided resonances on lithium niobate for extremelly small electric field detection investigated by accurate sensitivity analysis[END_REF]. This thin free-standing PCS can be fabricated from thin film Lithium Niobate-on-insulator (LNOI) wafers, which are nowadays commercially available thanks to ion slicing techniques [16]. The fabrication of the PCS on the LN suspended membrane has been assessed in a recent work [START_REF] Calero | Towards highly reliable, precise and reproducible fabrication of photonic crystal slabs on lithium niobate[END_REF],

demonstrating that highly precise and reproducible spectral performances are achievable.

However, the existence of an angle θ between the fiber facet and the PhC plane may produce a serious deterioration of the optical performances, strongly increasing the losses, as demonstrated in [START_REF] Calero | An ultra-side bandwidth, ultra-high spatial resolution and ultra compact electric field sensor based on Lab-on-fiber technology[END_REF]. Moreover, the macroscopic size of the ferrule makes even more critical the control of the parallelism since bigger airgaps, between the PCS and the fiber, are introduced when θ > 0, as depicted in Fig. 2(a). This non-parallelism angle can be minimized to negligible levels by replacing the rigid sample holder by a stretchable membrane. Thus, by applying a force, the membrane is bent, reducing the angle error as shown in Fig. 2(b). The applied force may break the 700nm-thick freestanding membrane. However, the rigid Si-substrate gives enough robustness to the chip to keep its integrity during the process. Once a strong force is applied to the stretchable membrane, both surfaces are forced to θ ≈ 0. Then, the epoxy glue can be applied to weld the ferrule and the TFLN which contains the PhC. The applied force on the ferrule towards the chip produces the necessary friction to avoid any displacement of the different elements that can be induced during the epoxy deposition. The epoxy is hardened during 3 hours at room temperature and then the ferrule is pulled back, leading to the assembly sketched in Fig. 2(d). The TFLN is directly in contact with the ferrule surface in order to minimize the air-gap between both surfaces that might lead to Fabry-Perot effect that can deteriorate the spectral performances. As sketched in Fig. 2(d), the chip diameter is slightly higher than the ferrule diameter in order to avoid the epoxy to be deposited on the PhC side. However, issues such as aligning both fiber and PhC centers or ensuring the optimal optical response are not considered in the standalone approach. For this reason, we assist the stretchable membrane approach by an active positioning system as further described.

The Assembling system

The experimental system which fulfills the mentioned requirements is represented in Fig. 3.

The system employs a supercontinuum source which allows a broadband characterization of the transmission spectrum during the assembly. In order to measure the transmitted spectrum, the light is firstly polarized in free space as the photonic crystal structure presents different Fano resonances as function of the incident polarization. Then, the laser is introduced into an inverted microscope which, using an 20x optical objective, injects the light precisely into the PhC structure. The alignment process requires the observation of the laser beam with respect to the PhC surface. For this reason, a VIR/NIS is introduced next to the objective to perform a first coarse alignment. The sample is, as previously described, located on a stretchable membrane. To avoid any perturbation to the microscope observation, the membrane presents a hole at its center. At this point, the ferrule can be approached by employing a 3-axes nano-precision piezoelectric to an approximate distance of 1μm away from the PhC. The ferrule can be firstly aligned by introducing any VIS/NIR light into the fiber, which transmits through the PhC and is captured by the camera. Once aligned, the supercontinuum light is collected by the ferrule and monitored by an Optical Spectrum Analyzer (OSA). The transmission at this distance is shown by the blue curve in Fig. 4(a), which is normalized with respect to the transmitted intensity of the cross polarization A sub-micrometer precision alignment is performed by optimizing both transmitted power and spectral extinction ratio as a function only of the x and y axes, reducing the degrees of freedom to the translation of x and y. Then, the epoxy is applied with an adjustable pipette. After 1 hour of curing, both trans-mission and reflection spectra are characterized. The resulting transmission spectrum from the assembly, represented by the red curve in Fig. 4(a), reveals that the spectral slope remains above 0.6dB/nm and does not degrade after the assembly. However, a shift of the resonant wavelength of approximately 5 nm is observed between both curves which can be attributed to the correction of the angle θ that changes the light incidence angle into the PhC [START_REF] Chen | Polarization and angular dependent transmission on transferred nanomembrane Fano filters[END_REF]. In order to obtain the reflection measurement, the supercontinuum source is plugged into the optical circulator. Since the PhC structure is polarization sensitive, the light injected into the ferrule has to be linearly polarized and aligned with the PhC structure. For this reason, a fiber stretching system is installed prior to the optical circulator. A feedback loop for the polarization control can be set by inserting a linear polarization analyzer to the camera, whose axis is aligned with respect to the PhC. The reflection spectrum is represented by the red curve in Fig. 4(b). It can be observed that both curves are complementary.The assembled sensor head is depicted in Fig. 5(a). The photonic chip, the fiber ferrule and the fiber can be clearly distinguished. A SEM picture of the photonic chip, before its assembly onto the ferrule, is shown in Fig. 5(e). As previously described, most of the chip contains the 370μm-thick Si substrate insuring robustness to the sensor head. Despite its circular shape, the chip presents a cut on a side in order to mark the orientation of the PhC and the LN crystallographic axes. At the center, the 250 μm-diameter free-standing membrane can be identified due to its darker tone, produced by the lower reflection of the SEM electrons induced by its ultra-thin thickness in comparison with the non-released areas. The membrane is zoomed in Fig. 5(c), where the PhC, whose sectional cut view shown in Fig. 5(d), is located at the center of the membrane. We notice that a bright square can be distinguished which corresponds to a drift-correction mark employed during the FIB fabrication.

Electric field sensing measurements

Due to the electro-optic (EO) properties of LN, any applied electric field along the crystallographic Z-axis will induce a linear modification of the refractive index with respect to the electric field. This modification leads to a shift on the resonant frequency of the Fano resonance. Thus, a continuous wave (CW) source has to be employed, whose wavelength is located within the Fano spectral slope. The CW light is then modulated by the applied E-field, whose modulation strength can be optimized by setting the wavelength at the maximum spectral slope of the Fano resonance [START_REF] Calero | Towards highly reliable, precise and reproducible fabrication of photonic crystal slabs on lithium niobate[END_REF]. In order to evaluate the EO performances, the fibered sensor is approached to a PCB (printed circuit board) containing two coplanar transmission lines where a time-variant periodic signal is applied as depicted in Fig. 6(a). The tunable source wavelength is set at 1576 nm ensuring the optimal EO modulation strength and linearity with respect to the applied E-field [START_REF] Calero | An ultra-side bandwidth, ultra-high spatial resolution and ultra compact electric field sensor based on Lab-on-fiber technology[END_REF]. Thus, a time-variant periodic signal is applied on the coplanar lines. During this experiment, the distance between the PC and the electrodes is approximately 600 μm. In addition, the position along the x axis is set to be a at the center of the gap region between the two electrodes since, as determined through electrostatic Finite Element Method (FEM) simulations, the maximum E-field value is located in this region as depicted in Fig. 6(b). The frequency of the introduced signal is set to 178 kHz due to an optimization of crosstalk interferences with other equipment surrounding the system. Moreover, the parameters of the network analyzer have been optimized to minimize the noise floor which has been estimated to be about -132.5 dBm. The reflected light, which carries the modulation signal, is introduced in a fast photodiode. The detected signal is split into high and DC signals by using its respective electrical filters. The DC signal carries the information about the reflected power in the absence of any E-field. This is useful since other factors such as a temperature change or deformation of the structure may add a shift of the resonant wavelength, displacing the optimal operation point. Thus, by monitoring the DC signal, the stability of the Fano sensor during its operation can be observed.

On the other side, the RF signal carries the E-field information. Thus, the signal is collected in a network analyzer whose measured bandwidth is represented in the inset of Fig. 6(c). The measured modulation frequency (f = 178 kHz) corresponds to the one at the frequency synthesizer and it exhibits an EO modulation strength greater than 30 dB with respect to the noise floor. The linearity of the device as a function of the E-field strength is assessed by varying its value from 40 V/m to 4 kV/m. The resulting EO modulation strength is represented in Fig. 6(b) in function of the applied E-field. A linear regression fit demonstrates a notable linearity, revealing the absence of non-linear effects strong enough to distort the linear operation regime. However, under 250 V/m the regression error increases probably due to the noise. Furthermore, the intersection between the noise floor and the linear regression determines the minimum detectable field whose value is estimated to approximately 170 V/m.

Conclusions

We have developed a procedure for the assembly of PhC structures on the tip of an optical fiber.

The technique presents several advantages with respect to other solutions. First, the stretching membrane simplifies the alignment procedure and decreases the alignment degrees of freedom from 5 to 2. Secondly, the technique includes dynamic positioning features allowing the observation of the photonic crystal optical response during the x-y axis optimization, allowing a sub-micrometer precision. This solution can be also employed for the integration of LN thin layers, also as well as for a broad range of materials that provide different functions such as temperature, pressure and media sensing. Furthermore, the technique requires relatively inexpensive equipment and may provide high throughput being compatible for batch process for the future industrialization of lab-on-fiber sensors.

Funding. This work was supported by the French Directorate General of Armaments (DGA) (ANR-13-ASTR-0019).

Fig. 1 .

 1 Fig. 1. Rendered images of (a) the fully assembled fibered sensor head and (b) sectional cut.

Fig. 2 .

 2 Fig. 2. Assembly of the ferrule-PCS: (a) Fiber approach to the PCS. (b) Pushing the fiber towards the stretchable membrane. (c) Epoxy deposition. (d) Fiber-tips sensor release.

Fig. 3 .

 3 Fig. 3. Experimental system including the dynamic positioning features.

Fig. 4 .

 4 Fig. 4. (a) Normalized transmission spectra for the free-standing PhC (blue) and for the assembled PCS (red). (b) Comparison of both transmission (blue) and reflection (red) of the assembled PCS.

Fig. 5 .

 5 Fig. 5. (a) Assembled sensor. (b) SEM picture of the sectional cut in a border of the membrane. (c) Top view of the free-standing membrane with the PhC structure in the middle. (d) PhC structure on the free-standing membrane. (e) SEM image of the chip with the membrane and the PhC

Figure 6 :

 6 Figure 6: (a) System for the measurement of the E-field sensing. (b) Spatial distribution of the E-field induced by electrodes. (c) Experimental variations of the EO modulation with respect to the applied E-field.

Acknowledgments. We appreciate the technical support of R.-M. Sauvage, L. Robert, S. Bargiel, T. Faure, E. Atie and R. Zegari. We are also grateful to the RENATECH network and the computing center of Franche-Comte.

Disclosures. The author declares no conflicts of interest.