Ph.D Yasmine Lamri

Shamila Vibhushan

Emeline Pacreau

Erwan Boedec

Ph.D Fanny Saidoune

Ph.D Arnaud Mailleux

M.D./ Bruno Crestani

Ph.D Ulrich Blank

Ph.D Marc Benhamou

M.D./ Thomas Papo

M.D./ Eric Daugas

M.D./ Karim Sacré

Ph.D Nicolas Charles
email: nicolas.charles@inserm.fr

Basophils and IgE contribute to Mixed Connective Tissue Disease development

Keywords: Basophil, IgE, Mixed Connective Tissue Disease, autoimmune disease pathophysiology, MCTD-like mouse model, interstitial lung disease C-C motif chemokine Receptor 3 chemokine receptor Ligand 11 CIC: Circulating Immune Complexes

Key messages

• MCTD patients have activated basophils and autoreactive IgE to U1-snRNP • Basophil depletion dampens lung pathology in a MCTD-like mouse model.

• IgE deficiency prevents the development of lung pathology in a MCTD-like mouse model.

• Basophils and IgE are promising therapeutic targets for Sharp Syndrome

INTRODUCTION

Mixed Connective Tissue Disease (MCTD) (or Sharp Syndrome) (1) is a rare systemic autoimmune disease (incidence around 2 per 100,000 adults in MN, USA) affecting mainly women (90%) [START_REF] Ungprasert | Epidemiology of Mixed Connective Tissue Disease, 1985-2014: A Population-Based Study[END_REF], whose clinical manifestations can overlap with other connective tissue diseases (CTDs) such as systemic lupus erythematosus (SLE), systemic sclerosis or myositis. Over 70% of MCTD patients have pulmonary involvement that may engage vital prognosis when diagnosed [START_REF] Venables | Mixed connective tissue disease[END_REF]. Serological hallmark of MCTD is the presence of high titers of autoreactive antibodies (autoAb) recognizing the 70 kDa subunit of the U1 small nuclear ribonucleoprotein (U1-snRNP 70k) along with the nearly complete absence of autoAb to double stranded DNA (dsDNA) or to Smith antigen (Sm), which are hallmarks of SLE [START_REF] Sapkota | Mixed Connective Tissue Disease[END_REF]. Although MCTD pathophysiology is not fully understood, post-translational modifications of U1-snRNP 70k are known to generate pathogenic neo-epitopes [START_REF] Hof | Autoantibodies specific for apoptotic U1-70K are superior serological markers for mixed connective tissue disease[END_REF]. This results in the accumulation of autoreactive B and T cells (mainly CD4 + ) recognizing U1-snRNP 70k and subsequent autoAb production [START_REF] Greidinger | CD4+ T cells target epitopes residing within the RNA-binding domain of the U1-70-kDa small nuclear ribonucleoprotein autoantigen and have restricted TCR diversity in an HLA-DR4-transgenic murine model of mixed connective tissue disease[END_REF]. Endothelial and immune cell activation via innate immune receptors, like Toll-Like Receptors (TLRs), Fc receptors and complement receptors by pathogenic complexes made of anti-U1snRNP antibodies and their antigen leads to vascular disease pathogenesis and tissue injury [START_REF] Enomoto | Bronchoalveolar lavage fluid cells in mixed connective tissue disease[END_REF][START_REF] Paradowska-Gorycka | U1-RNP and TLR receptors in the pathogenesis of mixed connective tissue diseasePart I. The U1-RNP complex and its biological significance in the pathogenesis of mixed connective tissue disease[END_REF][START_REF] Greidinger | A major B cell epitope present on the apoptotic but not the intact form of the U1-70-kDa ribonucleoprotein autoantigen[END_REF][START_REF] Okawa-Takatsuji | Endothelial cell-binding activity of anti-U1-ribonucleoprotein antibodies in patients with connective tissue diseases[END_REF].

Another evidence for the pathogenicity of the anti-U1snRNP antibodies comes from a mouse model that consists of immunizing mice against Human U1-snRNP and in which immunized mice develop a MCTD-like lung disease without any lupus-like features [START_REF] Greidinger | A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen[END_REF].

Being an autoimmune disease more frequent than MCTD, SLE has a better defined pathophysiology.

In SLE, autoreactive B and T cells accumulate in secondary lymphoid organs (SLO) and lead to the production of autoAb against multiple nuclear antigens (dsDNA, RNP, Sm, Ro, La). These autoAb aggregate with autoantigens and complement factors to form circulating immune complexes (CIC) that deposit in targeted organs such as joints, skin, central nervous system and/or kidney to induce an inflammation that may lead to organ failure. SLE evolving by relapse/remission cycles, flares can be triggered by innate immune signals such as viral infection or sun exposure. These triggers, along with CIC, can activate innate immune cells such as plasmacytoid dendritic cells, monocytes, 5 macrophages or neutrophils to amplify autoAb production through either cytokine production (type I interferon, B cell activating factor or BAFF…) or increased autoantigen availability (neutrophil extracellular traps or NETs) [START_REF] Dema | Advances in mechanisms of systemic lupus erythematosus[END_REF][START_REF] Kaul | Systemic lupus erythematosus[END_REF]. Interestingly, the mechanistic proximity of SLE with MCTD has been exposed in animal models. Indeed, Greidinger et al. demonstrated in their mouse model of MCTD that engagement of TLR3 and/or TLR7 could trigger the development of MCTD and/or SLE, respectively [START_REF] Greidinger | A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen[END_REF], revealing both kinship and mechanistic differences in their respective developments.

Patients with SLE or MCTD lack specific treatments and acute phases of these diseases are contained with high dose of immunosuppressive drugs and corticosteroids [START_REF] Venables | Mixed connective tissue disease[END_REF][START_REF] Sapkota | Mixed Connective Tissue Disease[END_REF][START_REF] Dema | Advances in mechanisms of systemic lupus erythematosus[END_REF][START_REF] Fanouriakis | update of the EULAR recommendations for the management of systemic lupus erythematosus[END_REF]. Recent development of immune cell targeting immunotherapies showed limited efficacy over the standards of care [START_REF] Fanouriakis | update of the EULAR recommendations for the management of systemic lupus erythematosus[END_REF]. The clinical needs prompt further research on the pathophysiology of these CTDs to identify specific targets that may allow reducing the adverse effects of the current therapeutic modalities.

Basophils are rare innate immune cells (<1% of peripheral blood leucocytes) known for their involvement in some allergic and parasitic diseases. Recent studies evidenced their immunoregulatory role in some type 2 immunity processes, especially as primary IL-4 producers [START_REF] Hussain | Basophil-derived IL-4 promotes epicutaneous antigen sensitization concomitant with the development of food allergy[END_REF][START_REF] Shibata | Basophils trigger emphysema development in a murine model of COPD through IL-4-mediated generation of MMP-12producing macrophages[END_REF][START_REF] Yamanishi | Basophil-derived IL-4 plays versatile roles in immunity[END_REF]. We demonstrated their IgE-and IL-4-dependent contribution to the development of SLE, especially of lupus nephritis, in both SLE patients and murine lupus-like models [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Dema | Basophils contribute to pristane-induced Lupus-like nephritis model[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. Basophils contribute to SLE pathophysiology by accumulating in secondary lymphoid organs (SLO) in a prostaglandin D2 (PGD2) and CXCL12 dependent manner [START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. Indeed, basophils from SLE patients and from two distinct Lupus-like mouse models overexpress PGD2 receptors (PTGDR-1 and PTGDR-2) and CXCR4, the receptor for CXCL12 [START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. Once recruited in SLO, basophils support short-lived plasma cell number and function, amplifying the peripheral titers of autoAb and CIC and their subsequent pathogenic effects [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Dema | Basophils contribute to pristane-induced Lupus-like nephritis model[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF].

Our previous studies on the contribution of basophils to SLE pathogenesis led to the identification of promising specific therapeutic targets in SLE, namely basophils themselves, PTGDR-1 6 and PTGDR-2 and IgE [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Dema | Basophils contribute to pristane-induced Lupus-like nephritis model[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF][START_REF] Dema | Immunoglobulin E plays an immunoregulatory role in lupus[END_REF]. The latter target showed promising effects in a human clinical trial that aimed at verifying the safety and tolerability of the anti-IgE humanized monoclonal antibody Omalizumab in SLE patients [START_REF] Hasni | Safety and Tolerability of Omalizumab: A Randomized Clinical Trial of Humanized Anti-IgE Monoclonal Antibody in Systemic Lupus Erythematosus[END_REF]. Since SLE and MCTD may be related in terms of pathophysiology, we sought to investigate the putative contribution of basophils and IgE into MCTD development.

Here, we report that basophils from MCTD patients have an activated phenotype, sharing some features with basophils from SLE patients but exhibiting specific increased markers such as C-C chemokine receptor 3 (CCR3) or unchanged expression of surface markers such as CD62L. The same basophil phenotype was observed in a MCTD-like mouse model, in which activated basophils accumulated in lungs and lymph nodes. U1-snRNP immunization-induced lung disease development was dependent on both basophils and IgE demonstrating their contribution to MCTD pathophysiology in this MCTD-like mouse model. Basophils played a central effector role in maintaining the lung pro-inflammatory environment allowing interstitial lung disease and fibrosis progressions in an IgE-dependent manner. Altogether, our study identifies basophils, IgE and pathways involved in their recruitment to the lungs as candidates for the investigation of new therapeutic modalities for MCTD patients.

METHODS

Patients

All Mixed Connective Tissue Disease (MCTD) patients fulfilled the accepted diagnostic criteria without fulfillment of classification criteria for SLE, systemic sclerodermia, idiopathic inflammatory myositis or rheumatoid arthritis [START_REF] Ungprasert | Epidemiology of Mixed Connective Tissue Disease, 1985-2014: A Population-Based Study[END_REF]. MCTD patients and healthy control (CT) donor characteristics are shown in Table E1 in the Online Repository. Blood samples were collected from adult patients enrolled in a prospective long term study of autoimmune diseases. The study was approved by the Comité Régional de Protection des Personnes (CRPP, Paris, France) under the reference ID-RCB 2014-A00809-38. MCTD patient samples were obtained from in-and outpatients and clinical data were harvested after approval by the Comission Nationale de l'Informatique et des Libertés (CNIL). A written informed consent was obtained from all individuals. All samples were collected in heparin blood collection tubes (Becton Dickinson) and processed within 4 hours as previously described [START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF].

Flow cytometry and antibodies 8

Antibodies used in both Human and murine flow cytometry data acquisition are presented in Table E2 (online repository). FACS data were collected with a LSRII-Fortessa flow cytometer using DIVA software (BD Biosciences) and analyzed with FlowJo software v10.0.7 (Treestar).

Antigen preparation

The DNA coding for U1-snRNP AA 63-205 was optimized for E. coli production and synthesized (Eurofins genomics). The sequence was amplified by PCR and introduced in the plasmids pET28b(+) (Novagen, Millipore SAS) and pMAL-C5X (New England Biolabs) by homologous recombination (GeneArt Seamless Enzyme Mix, ThermoFisher Scientific) and transformed into E. coli Top10 (ThermoFisher Scientific). pET28b(+) allowed the production of the 70K U1-snRNP AA 63-205 tagged by 6 histidine residues in N-terminal (His-tag). The resulting protein was used for anti-U1snRNP IgG and IgE ELISA (see below). pMAL-C5X allowed the production of the 70K U1-snRNP AA 63-205 fused to Maltose Binding Protein (MBP) in N-terminal of the fusion protein. The resulting plasmid pET28b-His-U1-snRNP and pMAL-C5X-MBP-U1-snRNP were sequenced and transformed into T7 Express Lys Y Competent E. coli (Lys Y E. coli) (New England Biolabs). Lys Y E. coli was growth at 37°C in LB-media supplemented with Kanamycin (50µg/mL) for pET28b plasmid and carbenicylin (100 µg/mL) and glucose (2 g/L) for pMAL-C5X. The productions of both recombinant proteins were induced by β-d-1thiogalacto-pyranoside (IPTG) at 0.5 mM final during mid-exponential phase and incubated overnight at 27°C. Bacteria were pelleted and lysed by sonication. Supernatants were collected and purified on HiTrap™ TALON crude column for pET28b plasmid and MBPTrap™ HP column for pMAL-C5X plasmid (both from GE Healthcare Life Sciences). The production of MBP alone was performed with the empty vector pMAL-C5X and followed the same production protocol than pMAL-C5X-MBP-U1-snRNP. Purity of produced recombinant protein was assessed by SDS-PAGE and Coomassie blue staining and concentrations were determined by BCA protein array (ThermoFisher Scientific).

Mixed Connective Tissue Disease-like mouse model

9 At day 0, each mouse received subcutaneously (sc) 100 µL of a solution of complete Freund Adjuvant (CFA) emulsified vol./vol. with 50 µg of MBP-U1-snRNP (70k) fusion protein or MBP alone diluted in PBS (ThermoFisher Scientific) in the interscapular region. At day 14, mice received a sc injection of the same dose/antigen as on day 0 but emulsified in incomplete Freund adjuvant (IFA) vol./vol. Our MCTD-like induction protocol was adapted from the one from Greidinger et al. [START_REF] Greidinger | A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen[END_REF] and led 100 % of mice immunized against MBP-70K-U1-snRNP to develop both antibodies to these proteins and the expected MCTD-like lung disease. Mice were euthanized and analyzed at 6 weeks post-immunization as illustrated in FIG 2A.

Enzyme linked Immunosorbent Assays (ELISA)

Levels of IgG and IgE anti-U1-snRNP in human plasma or mice were measured by semi-quantitative ELISA on plates coated with the Human U1-snRNP protein His-tag (see antigen preparation section) at 1 µg/mL in phosphate buffered saline (PBS). The detection antibodies used were horseradish peroxidase (HRP)-conjugated sheep anti-mouse-IgG (Jackson ImmunoResearch laboratories), goat anti-mouse IgE (Bethyl laboratories), goat-anti-Human IgG (Bethyl laboratories) or goat anti-Human IgE (Bethyl laboratories) following manufacturer's instructions. Total IgE levels in patient and healthy control plasma samples were measured with the goat anti-Human IgE ELISA kit (Bethyl laboratories) following manufacturer's instructions.

Mouse sample processing

Mice were euthanized by CO2 inhalation and processed as previously described [START_REF] Dema | Basophils contribute to pristane-induced Lupus-like nephritis model[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. For lung analysis, Broncho Alveolar Lavage (BAL) fluid was obtained by cannulating the trachea with a 20gauge needle via an incision, and instilling 3x1 mL of Phosphate Buffered Saline (PBS). The whole lungs were lavaged and approximately 2.5 mL of BAL fluid was retrieved and centrifuged at 450 g for 5 min before resuspending the cell pellet in FACS buffer (PBS containing 1 % Bovine Serum Albumin and 0.05% NaN3). After this step, mice were perfused with 25 mL of PBS and the whole perfused lung was isolated. The left lobe was ligated and inflated with 10% of formalin (Sigma Aldrich) during 24 10 hours. The lung tissues were embedded in paraffin and 5 µm sections were prepared, followed by a Masson's trichrome staining or kept for immunohistochemistry staining. The lobes of the right lung were separated for a specific use. Middle lobes were stored at -80°C in RNAprotect Tissue Reagent (Qiagen) for later RNA extraction and RT-qPCR analysis. The inferior lobe were digested with 0.2 mg/mL of Liberase TM (Roche), 0.1 mg/mL of DNAse (Sigma-Aldrich) in 1mL of RPMI 1640 containing 1% of Fetal Calf Serum (Gibco) for 1hr at 37 °C and then filtered using a cell strainer (40 µm) (Corning). The homogenate was washed in FACS buffer, centrifuge at 450 g for 5min before surface staining for flow cytometry analysis.

Immunohistochemistry

Histological sections of the lungs were dewaxed with xylene, rehydrated in graded concentrations of ethanol, and washed with PBS. Blocking of non-specific binding was realized by 30 minutes incubation in PBS containing 5% of goat serum. Antigen retrieval was performed by heating the slides 1x2minutes then 2x5minutes in Citric Buffer (10 mM, pH=6) using a microwave. Then, the anti-Rat HRP Cell & Tissue Staining Kit (R&D systems) was used following the manufacturer's instructions and with rat anti-mouse MCPT8 Antibody (clone TUG8, BioLegend) or isotype control rat IgG2a,κ (BioLegend) diluted in PBS containing 5% of goat serum to a final concentration of 1.5 µg/mL. To amplify the signal, sections were incubated with an Avidin-Biotin peroxidase complex (VECTASTAIN® ABC-HRP kit, Vector laboratories). MCPT8 localization was then visualized with a peroxidase substrate using the ImmPACT® DAB substrate diluent (Vector laboratories). Sections were then dehydrated with gradually increasing concentrations of Ethanol and then placed in xylene. Finally, the sections were counterstained with hematoxylin and coverslipped using the Eukitt® mounting medium (Kisker Biotech).

Lung pathology scores

A score of the pathology was designed to quantify inflammatory cell infiltrations in the interstitial, peribronchial and perivascular areas of the lungs. 5 µm thin sections were stained with Masson's 11 trichrome. Stained slides were scanned with an Aperio CS2 digital pathology slide scanner (Leica) and analyzed with the Aperio ImageScope software v.12.3 (Leica). For each area, 10 fields per lung as depicted in Figure 2 were scored from 0 to 3 as follows: 0: absence of infiltrate; 1: low number of infiltrating cells (5-20 % of the observed area); 2: moderate number of infiltrating cells (25-50 % of the observed area); 3: high number of infiltrating cells (more than 50 % of the observed area)). The mean score per affected area was calculated and multiplied by the proportion of fields showing affected areas.

Quantitative RT-PCR

RNA extraction was performed as described in the manufacturer's protocol (RNeasy Mini kit, Qiagen). cDNA was synthesized with iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed with SsoAdvanced SYBR green reaction mix (Bio-Rad) using the following KiCqStart TM Primers pairs (purchased from Sigma-Aldrich): M_Il4_1; M_Ccl11_1; M_Ifng_1; M_Actb_1;

In-vivo basophil depletion

13

RESULTS

MCTD patients have activated basophils and autoreactive IgE raised against U1-snRNP

Basophils contribute to SLE pathogenesis during which they show an activated phenotype, as we previously described [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. MCTD having a SLE-related pathophysiology, we analyzed the phenotype of peripheral blood basophils from a small prospective cohort of MCTD patients and compared these basophil phenotypes to healthy volunteers (CT) matched for sex and age, whose Repository). These data suggested that basophils do not have the same behavior in this MCTD-like mouse model as compared to SLE-like mouse models where they accumulate in all secondary lymphoid organs (spleen and lymph nodes) and not in the injured organ (kidney) [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. This suggested that basophils may be involved in the development of the lung pathology observed in the MCTD-like mouse model, both at the humoral level (through lymph node accumulation) and probably at the effector level through lung accumulation.

Basophils promote MCTD-like lung disease

In order to evaluate basophil contribution to the development of the lung pathology in this MCTDlike mouse model, we depleted basophils through the injection of diphtheria toxin (DT) during the last 10 days of the protocol (see Methods and FIG 2A). We then evaluated the lung pathology, immune cell infiltrates in the lungs and collagen and pro-inflammatory cytokines at the mRNA level. Altogether, these data demonstrate that basophil depletion in the MCTD-like mouse model used dampened the measured lung pathology in terms of interstitial, peribronchial and pervivascular cellular infiltrates, CD4 + T cell accumulation and pro-inflammatory and pro-fibrotic environment.

IgE deficiency prevents the activation of basophils during MCTD-like disease development

IgE deficiency prevents MCTD-like lung disease development

Based on this, we next evaluated the MCTD-like lung pathology in the IgE-deficient mice (Igh7 -/-).

Like basophil depletion (FIG

DISCUSSION

Current understanding of MCTD pathophysiology is limited. Autoreactive T and B cells cooperate to drive the production of pathogenic autoantibodies and immune complexes that target endothelial cells and putatively specifically, at least in a majority of patients, the lung tissue. As for a number of autoimmune diseases, including connective tissue diseases such as SLE, MCTD patients have no specific treatment available. Thus, developing knowledge about cells and mediators involved in disease development, tissue injury and organ function loss will benefit affected patients to conceive new therapies for the studied disease. Moreover, for distinct clinical entities that share some pathophysiological processes, developing (or repurposing) innovative therapeutic modalities for one given disease may benefit also to all the related conditions.

We previously described the contribution of basophils, IgE and autoreactive IgE to the pathogenesis of SLE [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Dema | Basophils contribute to pristane-induced Lupus-like nephritis model[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF][START_REF] Dema | Immunoglobulin E plays an immunoregulatory role in lupus[END_REF][START_REF] Dema | Autoreactive IgE is prevalent in systemic lupus erythematosus and is associated with increased disease activity and nephritis[END_REF]. Our studies allowed to decipher the mechanisms by which basophils are activated and accumulate in secondary lymphoid organs in various SLE-like mouse models and more importantly in SLE patients [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Dema | Basophils contribute to pristane-induced Lupus-like nephritis model[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. These studies, which were confirmed by other laboratories [START_REF] Henault | Self-reactive IgE exacerbates interferon responses associated with autoimmunity[END_REF][START_REF] Pan | Basophil Activation-Dependent Autoantibody and Interleukin-17 Production Exacerbate Systemic Lupus Erythematosus[END_REF], led us to test new therapeutic modalities for SLE patients and led to a phase Ib clinical trial evaluating the safety and tolerability of Omalizumab (a humanized anti-IgE monoclonal antibody) in SLE patients that showed some promising preliminary results [START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF][START_REF] Hasni | Safety and Tolerability of Omalizumab: A Randomized Clinical Trial of Humanized Anti-IgE Monoclonal Antibody in Systemic Lupus Erythematosus[END_REF]. Here, we demonstrate that both basophils and IgE may as well represent relevant therapeutic targets in the context of Sharp syndrome. Indeed, basophil depletion dampened lung cellular infiltrates, reduced pro-TH2 and proinflammatory environment and prevented fibrosis initiation in the lungs in the MCTD-like mouse model. Similarly, IgE deficiency completely prevented disease development. In that perspective, the present study identifies both basophils and IgE as new therapeutic targets in MCTD.

The prevalence of autoreactive anti-U1-snRNP (70) IgE in our cohort was around 80%. The analysis of a larger number of patients in the coming years will allow to determine a more precise prevalence of 19 such autoreactive IgE and whether other autoantigen specificities for IgE can be detected in MCTD patients.

Basophil depletion in our study was achieved by using the Mcpt8 DTR mice on the C57BL/6J genetic background developed by Wada et al. [START_REF] Wada | Selective ablation of basophils in mice reveals their nonredundant role in acquired immunity against ticks[END_REF]. Recently, El Hachem et al. nicely reported that injections of a similar dose of DT was inducing granulocyte-macrophage progenitor depletion in the bone marrow of Mcpt8 DTR mice on the Balb/c genetic background, resulting in a depletion of eosinophils and neutrophils [START_REF] Hachem | Treatment of MCPT8(DTR) mice with high-or low-dose diphtheria toxin leads to differential depletion of basophils and granulocytemacrophage progenitors[END_REF]. In our set up, 250 ng of DT per injection could not achieve more than a 50% depletion of basophils (data not shown). The use of 1 µg DT per injection was mandatory to achieve basophil complete depletion and did not lead to the depletion of eosinophils or neutrophils in all the analyzed compartments (FIG E4 and E5 in the Online Repository). This is more than likely due to a difference in the bioactivity of the unnicked DT used and/or to the different genetic backgrounds of the mice.

The MCTD-mouse model used here, adapted from the original model developed by Greidinger et al. [START_REF] Greidinger | A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen[END_REF], involves an immunization with complete Freund adjuvant followed by a boost in incomplete Freund adjuvant. Thus, the present study could not evaluate the early immunoregulatory role of basophils in the production of autoreactive antibodies and in its amplification as we did in the SLE context [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. Indeed, basophil depletion did not impact the titers of anti-U1-snRNP antibodies in immunized mice (FIG 2) as expected since basophils are not required for CFA-induced antigen sensitization in vivo [START_REF] Ishida | Adjuvants determine the contribution of basophils to antigen sensitization in vivo[END_REF]. However, based on our previous studies and on the phenotype of MCTD patient basophils (CXCR4 increased expression), these cells, by accumulating in lymph nodes, may support autoantibody production as they do in SLE. A spontaneous MCTD-like mouse model is still lacking to the field to decipher further the first steps of the MCTD pathophysiology. Another limitation of the MCTD-like mouse model used in our study is the time chosen for analysis. On one hand, it allowed describing the high relevance of basophil effector function in the primary inflammatory processes of the lung disease development. On the other hand, later time points of the 20 disease should be analyzed as well, once the lung fibrosis is well established and basophil contribution to such late phases should be addressed. Indeed, interstitial lung disease in patients is often diagnosed once it is well established and rarely at the very early phases. Thus, evaluating basophil depletion as a putative therapeutic approach in lungs more impacted by fibrosis may be of interest for translation to human therapeutics.

Targeting directly basophils may then be of clinical interest in MCTD and SLE as suggested by our results. A therapy targeting specifically basophils at the cellular level is not, to our knowledge, currently available. However, a promising anti-CD123 (the IL-3 receptor α chain) monoclonal antibody, CSL362, allows targeting key cells in CTD pathogenesis which are plasmacytoid dendritic cells and basophils [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF][START_REF] Henault | Self-reactive IgE exacerbates interferon responses associated with autoimmunity[END_REF][START_REF] Oon | A cytotoxic anti-IL-3Ralpha antibody targets key cells and cytokines implicated in systemic lupus erythematosus[END_REF][START_REF] Oon | A potential association between IL-3 and type I and III interferons in systemic lupus erythematosus[END_REF]. Interestingly, the anti-CD123 targeted cells are the same as the one activated by autoreactive IgE in SLE and MCTD [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Henault | Self-reactive IgE exacerbates interferon responses associated with autoimmunity[END_REF][START_REF] Pan | Basophil Activation-Dependent Autoantibody and Interleukin-17 Production Exacerbate Systemic Lupus Erythematosus[END_REF].

Concerning the mechanistic understanding of MCTD lung pathology, our report provides some new parameters to take into accounts in further studies. The most striking marker differentially expressed on MCTD patient basophils was CCR3, suggesting an involvement of basophil, CCR3 and its main ligand CCL11 in the course of the disease and this was recapitulated in the MCTD-like mouse model.

Despite the fact that the lung pathology at the time of analysis was really mild, we could detect collagen I mRNA accumulation in lungs of U1-snRNP immunized mice suggesting the early development of lung fibrosis. This parameter was clearly associated with the detection of IL-4 and CCL11 mRNA in the same lung extracts. Strikingly, basophil depletion or IgE deficiency completely normalized such pathological parameters and also dampened CD4+ T cells recruitment to the lungs. CCL11 in the lungs is induced by IL-4 and allows recruitment of CCR3 bearing cells in the bleomycininduced mouse model of lung fibrosis [START_REF] Huaux | Role of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosis[END_REF][START_REF] Hosokawa | IL-4 Modulates CCL11 and CCL20 Productions from IL-1beta-Stimulated Human Periodontal Ligament Cells[END_REF]. Recently, basophil-derived IL-4 was shown to promote emphysema in a mouse model of elastase-induced chronic obstructive pulmonary disease (COPD) [START_REF] Shibata | Basophils trigger emphysema development in a murine model of COPD through IL-4-mediated generation of MMP-12producing macrophages[END_REF]. Then, one working hypothesis could be that basophils, activated through anti-U1-snRNP antibodies in a complex with their autoantigen, and overexpressing CCR3 would tend to accumulate 21 in airways and lung tissue. Once recruited, their IL-4 production may upregulate CCL11 levels in the airways, leading to both recruitment of CCR3 expressing CD4 + TH2 cells and to the maintenance of IL-4 pressure to amplify TH2 differentiation. Such an amplification loop of the pro-fibrotic TH2 environment in the lungs may then facilitate and induce long-term development of lung fibrosis. In this scenario, basophils may then play both effector and immunoregulatory roles in the pathophysiology of MCTD lung disease development.

IgE deficiency completely prevented basophil activation and accumulation both in lymph nodes and lungs, suggesting that the role of basophils depends on IgE-mediated activation in the MCTD pathogenesis. A spontaneous mouse model of MCTD will be required to determine whether basophils are as well involved in the generation or the amplification of such autoreactive IgE and IgG productions and contribute to an amplification loop of the disease as they do in SLE.

Our study identifies the basophil/IgE/IL-4/CCL11 axis as a relevant candidate for further investigations aiming to define specific therapeutic approaches for lung involvement in Sharp Syndrome. For instance, antagonizing CCR3 and CCL11 interaction may be a valuable approach to prevent basophil accumulation in lungs and their deleterious effects. This approach should be selectively appreciated in MCTD and probably not in SLE, since in the latter condition, we could not detect any variation of CCR3 expression by basophils [START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF].

As suggested by our present report, targeting IgE to prevent the actions of autoreactive IgE may resume the beneficial effects that we observed on lung pathology in the context of IgE deficiency. As for SLE, Omalizumab may be a reasonable, safe and effective approach [START_REF] Hasni | Safety and Tolerability of Omalizumab: A Randomized Clinical Trial of Humanized Anti-IgE Monoclonal Antibody in Systemic Lupus Erythematosus[END_REF] along with other IgE targeting monoclonal antibodies such as ligelizumab and UB-221 that are currently under clinical development for treatment of chronic spontaneous urticaria, another autoimmune condition dependent on autoreactive IgE [START_REF] Kolkhir | New treatments for chronic urticaria[END_REF].

FIGURE LEGENDS

Mice 8 weeks

 8 old female Mcpt8 DTR mice (23) on a C57BL/6J genetic background bred in our pathogen-free animal facility were kindly provided by Prof. Hajime Karasuyama (Tokyo Medical and Dental University, Tokyo, Japan). 8 weeks old female Igh7 -/-mice (24) on a C57BL/6J genetic background bred in our pathogen-free animal facility were kindly provided by Prof. Hans Oettgen (Harvard Medical School, Boston, Massachusetts, USA) and Dr Juan Rivera (National Institute of Arthritis, Musculoskeletal and Skin diseases (NIAMS), NIH, Bethesda, MD, USA). Mice were maintained following the French and European guidelines and the study was approved by the local ethical committee (comité d'éthique en expérimentation animale, Faculté de Médecine Site Bichat Université Paris Diderot) and by the Department of Research of the French government under the animal study proposal number APAFIS# 14115.

  M_Col3a1_2; M_Col1a1_2 and M_Cxcl12_1 to respectively measure IL-4, CCL11, IFNγ, β-actin, COL3A1, COL1A1 and CXCL12 mRNA expression levels. Quantitative PCR was performed on the CFX96 Touch Real Time PCR Detection System (Bio-Rad) and following amplification, Ct values were obtained using the CFX Manager TM software 2.1 (Bio-Rad).

Female

  Mcpt8 DTR mice (with the C57BL/6 genetic background) received four intraperitoneal injections of 1 µg of diphteria toxin (D05664; Sigma-Aldrich) (or the corresponding vehicle, PBS) in 100 µL of PBS 10, 9, 4 and 1 days before sacrifice as illustrated in FIG 2A. Statistics Distribution was assessed with D'Agostino-Pearson omnibus normality test or Kolmogorov-Smirnov test, depending on sample size. When more than 2 groups were compared, one-way analysis of variance (ANOVA) tests were conducted before the indicated post-tests. All tests run were twotailed. Data are represented either as mean ± s.e.m. or as median and interquartile ranges with 12 whiskers representing 5-95 percentiles and the mean presented as a '+' symbol. In all figures, comparison to control group is shown above each bar and to the corresponding bars when indicated. Statistics were performed with GraphPad Prism version 6.0 software. NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. A.U.: arbitrary units.

  characteristics are described in TableE1(in the Online Repository). The flow cytometry gating strategy is described in FIG E1A (see the Online Repository). MCTD patient basophils showed an activated phenotype (FIG1A-G). Interestingly, the global activation profile of basophils from MCTD patients differed to some extent from that of SLE patients[START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Pellefigues | Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs[END_REF]. Indeed, like SLE patients, MCTD patients showed a peripheral basopenia (FIG 1A-B), an increased expression of the activation marker CD203c and of the CXCL12 chemokine receptor CXCR4 on their basophils when compared to CT (FIG 1C-D). Unlike SLE patients (18, 20), CD62L expression was not significantly increased on MCTD patient basophils whereas CCR3 (the chemokine receptor mainly for eotaxin (CCL11) and CCL5) expression was dramatically increased (FIG 1E-F). Since CCL11-CCR3 axis is known to drive granulocyte recruitment in lungs in allergic airway inflammation and in a mouse model of lung fibrosis (25, 26), this increased CCR3 expression along with peripheral basopenia may suggest a stronger lungoriented migration abilities of basophils in MCTD patients. A significant trend on increased expression of FcεRIα on the surface of MCTD patient basophils was also noticed (FIG 1G) but was not associated with significantly increased total IgE levels (FIG 1H) unlike what is observed in allergic or SLE patients (18, 27). The latter parameter suggested a qualitative over a quantitative serological difference concerning IgE. As expected, all MCTD patients had anti-U1 snRNP (70k) autoreactive IgG (FIG 1I) and nearly 80% of them had significant levels of anti-U1 snRNP (70k) autoreactive IgE (FIG 1J and Table E1 in the Online Repository) (significant defined as superior to mean of CT values + 2 standard deviations). 14 Altogether, these results indicated that basophils were activated in MCTD patient blood during the course of the disease, as hypothesized. However, their activation pattern and the expressed levels of the chemotactic receptors CCR3 was clearly different from the profiles observed on healthy donor and SLE patient basophils, suggesting specific migration abilities of basophils during MCTD. Moreover, autoreactive IgE raised against U1-snRNP may contribute to such basophil activation profile in MCTD.Basophils are activated and accumulate in lymph nodes and lungs in a MCTD-like mouse modelWe next sought to verify whether we could identify a basophil phenotype similar to the one observed in MCTD patients (FIG 1) in a modified version of a MCTD-like mouse model developed by Greidinger et al. (11). This model consists of immunizing mice against a fusion protein between the maltose-binding protein (MBP) and a portion of the Human 70kDa protein of the U1-snRNP complex as previously described (see Methods, FIG 2A and (11)). The control mice (Mcpt8 DTR mice on a C57BL6/J background) were immunized against the MBP alone allowing to rule out any effect of the immunization process per se. All immunized mice developed antibodies to MBP (FIG 2B), and all U1-snRNP-MBP immunized mice developed antibodies of IgG and IgE isotypes against U1-snRNP (FIG 2C-D). All mice from the latter group developed cellular infiltrates in the perivascular, peribronchial and interstitial areas of the lungs (FIG 2E and see below) accordingly to the original model (11). Of note, no sign of SLE-like disease was observed in the MCTD-like mice as no IgG-and C3-containing immune complex deposits were observed in their kidneys nor anti-dsDNA IgG in their blood (data not shown). These data indicate that the modified protocol of the MCTD-like model led to the expected immunization against the U1-snRNP antigen with anti-U1-snRNP antibodies of IgG and IgE isotypes, and to MCTD-like lung disease development without inducing a SLE-like phenotype. This modified model was then relevant to analyze basophil contribution to the disease. As for SLE-like mouse models (18-20), we could not recapitulate the peripheral basopenia observed in MCTD patient blood (FIG 1A-B and FIG 3A). However, blood basophils from U1-snRNP-MBP immunized mice showed a similar phenotype to the MCTD patient blood basophils with an increased activation status (CD200R expression), and increased CCR3 and CXCR4 levels (FIG 3B-D). Interestingly, basophils neither accumulated in the spleen of the MCTD-like mice nor in bone marrow (FIG E2 in the Online Repository), but did accumulate in the upper lymph nodes of the mice (cervical and brachial), in broncho-alveolar lavage (BAL) and in the lung tissue (FIG 3E-L and FIG E3-E5 in the Online

  Basophil depletion was effective and specific for the basophil compartment (FIG E4 and E5 in the Online Repository). The basophil depletion dampened the cellular infiltrates in peribronchial, perivascular and interstitial areas (FIG 4A-C), demonstrating basophil contribution to this mild MCTDlike lung pathology development. As the original MCTD-like mouse model, the modified model was characterized by a marked CD4 + T cell recruitment into the lungs ((6, 11), FIG 4D and FIG E5F in the Online Repository). U1-snRNP-MBP immunization and/or basophil depletion did not lead to any difference in the proportions of all other hematopoietic cell types analyzed in the lungs of the mice 16 (FIG E5 in the Online Repository). Accordingly and as expected (28, 29), 10 days long basophil depletion neither reduced anti-MBP nor anti-U1snRNP antibody titers (FIG 2A-D) in the sustained immunization protocol followed (see Methods, FIG 2A and (28)). Clearly, however, basophil depletion led to a dramatic decrease in the proportions of CD4 + T cells recruited to the lungs during the course of the disease (Fig. 4D and FIG E5F in the Online Repository) suggesting a central effector role of basophils for the accumulation of pathogenic CD4+ T cells in the MCTD-like lung disease development. Next, we analyzed the expression levels of various genes of interest in mRNA extracted from the lungs of immunized mice (FIG 4E-H and FIG E6 in the Online Repository). First, analysis of collagen I and III mRNA expressions demonstrated, as already shown at the morphology level (FIG 2E), that the induced lung disease at the time of sacrifice did not reach the fibrotic state as evidenced by unchanged Col3a1 mRNA levels (FIG 4E). However, the development of lung fibrosis was putatively initiated as increased levels of Col1a1 mRNA were detected (FIG 4F) along with increased IL-4 and CCL11 mRNA levels (Fig. 4G,H). In line with the reduced lung pathology observed in basophildepleted MCTD-like mice (FIG 4A-D), basophil depletion led to normalized levels of Col1a1, IL-4 and CCL11 mRNAs (FIG 4F-H), strongly suggesting that basophils were required to organize the pro-TH2 and pro-fibrotic inflammatory environment in the lungs during the course of the disease. Of note, basophil depletion also reduced the pro-TH1 inflammatory environment as evidenced by the significant reduction in IFNγ mRNA levels observed in the lung of the U1-snRNP-MBP immunized mice, whereas no change in CXCL12 mRNA levels were observed in any of the analyzed conditions (FIG E6 in the Online Repository).

17 As

 17 we observed the high prevalence of autoreactive IgE both in MCTD patients and in mice developing MCTD-like lung disease, we investigated the contribution of IgE in basophil activation and localization in lungs and lymph nodes during the development of the MCTD-like lung disease. For this purpose, we immunized IgE-deficient mice (Igh7 -/-(24)) with either MBP alone or U1-snRNP(70k)-MBP fusion protein and evaluated their basophil phenotype. As anticipated, IgE deficiency did not influence the proportion of blood basophils (FIG 5A) in the U1-snRNP70k-MBP immunized mice but completely prevented their activation (FIG 5B), their up-regulation of surface CCR3 and CXCR4 (FIG 5C-D) and their accumulation in lymph nodes (FIG 5E). This was accompanied by the nearly complete abrogation of basophil accumulation in the BAL of IgE-deficient mice immunized with the U1-snRNP-MBP (FIG 5F). These results strongly support the critical role of IgE in the activation of basophils during MCTD-like disease development enabling their migration to lymph nodes and accumulation in lungs.

4 ),

 4 IgE deficiency similarly protected the immunized mice from developing perivascular, peribronchial or interstitial immune cell infiltrates (FIG 6A-C) including the accumulation of CD4 + T cells in lungs (FIG 6D). Accordingly, no increase in Col3a1, Col1a1, IL-4 and CCL11 mRNA levels was observed in IgE-deficient mice immunized with U1-snRNP (70k)-MBP when compared to their MBP-immunized counterparts (FIG 6E-H). Importantly, immunization against either MBP or U1-snRNP-MBP antigens were as efficient in terms of specific IgG production in IgEsufficient and IgE-deficient animals (FIG E7 in the Online Repository). Altogether, these data demonstrate that IgE deficiency prevents basophil activation, their subsequent migration to lymph nodes and lungs, and the basophil-dependent development of the MCTD-like lung disease.

Figure 1 :

 1 Figure 1: MCTD patients have activated basophils and autoreactive IgE raised against U1-snRNP

Figure 2 :

 2 Figure 2: U1-snRNP-MBP immunization induces inflammatory infiltrates in lungs, and the

Figure 3 :

 3 Figure 3: Basophils are activated and accumulate in lymph nodes and lungs during MCTD-like

Figure 4 :

 4 Figure 4: Basophils contribute to MCTD-like lung disease development

Figure 5 :

 5 Figure 5: IgE deficiency abrogates basophil activation and accumulation in lymph nodes and lungs

Figure 6 .

 6 Figure 6. IgE deficiency prevents MCTD-like lung disease development

ACKNOWLEDGMENTS

The authors wish to acknowledge the nurses and clinicians from Bichat hospital's Internal Medicine, Nephrology and Pulmonology departments and all the members of the Centre de Recherche sur l'Inflammation for their invaluable help. We thank Pr Hajime Karasuyama from the Department of Immune Regulation, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan, for providing the Mcpt8 DTR mice. We also thank Prof.

Hans Oettgen (Harvard Medical School, Boston, MA, USA) and Dr Juan Rivera (National Institute of Arthritis, Musculoskeletal and Skin diseases (NIAMS), NIH, Bethesda, MD, USA) for generating and providing the Igh7 -/-mice, respectively. We are indebted to the colleagues from animal care, flow cytometry and morphology core facilities of the Bichat School of Medicine.

This work was funded by ANR-11-JSV1-009-01 BASILE, ANR-15-CE17-0002 BATTLE, ANR-PIA-INFLAMEX, CNRS, AP-HP and INSERM. PhD fellowship support for Yasmine Lamri was provided by the French ministry for higher education, research and innovation (MESRI).

 E1 andE2).