Maud Lacombe

Michel Jaquinod

Lucid Belmudes

Yohann Couté

Claire Ramus

Florence Combes

Thomas Burger

Elisabeth Mintz

Justine Barthelon

Vincent Leroy

Aurélia Poujois

Alain Lachaux

France Woimant

Virginie Brun

Comprehensive and comparative exploration of the Atp7b(-/-) mouse plasma proteome

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Introduction

Wilson's disease (WD) is a rare genetic disorder affecting approximately 1/30 000 new-borns; it is characterised by toxic copper accumulation in the liver and brain. WD is a monogenic autosomal-recessive condition resulting from mutations in the ATP7B gene located on chromosome 13 in humans. More than 600 different mutations of the ATP7B gene have been described, associated with reduced expression or functional impairment of ATP7B protein, a copper-transporting P-type ATPase essential to maintaining copper homeostasis. [START_REF] Poujois | Wilson's disease: A 2017 update[END_REF] This 164-kDa trans-membrane protein is expressed at high levels in hepatocytes in the trans-Golgi network where it delivers copper cofactor to cuproenzymes, such as ceruloplasmin, before they are secreted into plasma. In response to increased cellular copper concentrations, ATP7B translocates to the endolysosomal compartment and the plasma membrane of bile canaliculi to promote copper efflux into the bile. [START_REF] Hasan | Molecular events initiating exit of a copper-transporting ATPase ATP7B from the trans-Golgi network[END_REF] In WD, perturbation of ATP7B protein expression, stability, function, or intracellular targeting impair copper delivery to the secretory pathway and copper excretion into the bile. Disruption of copper homeostasis causes massive copper accumulation in hepatocytes, leading to irreversible oxidative damage and, ultimately, cell death. A massive release of free copper (unbound to ceruloplasmin) into the bloodstream is then observed which contributes to extending the free copper overload and toxicity throughout the body, particularly in the central nervous system. Although the genetic mutations causing WD are present even before birth, clinical symptoms usually only appear during childhood or teenage years. WD is primarily hepatic with a wide spectrum of manifestations, spanning from a slight (clinically undetectable) elevation of transaminases to acute liver failure, acute-on-chronic hepatitis, and cirrhosis. Neurological symptoms including tremors, dysarthria, dystonia, or Parkinsonian symptoms may dominate initially or appear gradually as the liver disease progresses. These neurological disorders are often associated with psychiatric disorders (depression, anxiety). [START_REF] Roberts | Diagnosis and treatment of Wilson disease: an update[END_REF] The "Kayser-Fleisher ring" is a specific ocular symptom of WD resulting from copper accumulation around the cornea; it is present in 95% of patients with neurological symptoms. [START_REF] Poujois | Wilson's disease: A 2017 update[END_REF] Therapeutic management of WD patients requires lifelong administration of copper-chelating agents and/or zinc salts to reduce intestinal copper absorption. Liver transplantation is necessary to treat fulminant hepatitis or decompensated liver cirrhosis. As the more serious symptoms appear later, earlier detection of WD would allow early initiation of pharmacological treatments, before patients develop hepatic and neurological diseases alongside irreversible organ damage. However, because of its low incidence, its variable clinical presentations, and the lack of a single 100% reliable diagnostic parameter that can exclude or confirm the diagnosis of WD, early detection remains challenging. WD is thus generally only confirmed following a combination of biological, ophthalmological (recognition of "Kayser-Fleisher ring"), brain MRI, and genetic tests. In combination with classical liver function tests, specific biological assays have been developed to detect decreased serum ceruloplasmin concentrations and increased urinary copper excretion. [START_REF] Poujois | Wilson's disease: A 2017 update[END_REF][START_REF] Roberts | Diagnosis and treatment of Wilson disease: an update[END_REF] Recently, assays measuring exchangeable serum copper and relative exchangeable copper were also proposed to improve WD diagnosis. [START_REF] Balkhi | Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis[END_REF][START_REF] Heissat | Evaluation of the accuracy of exchangeable copper and relative exchangeable copper (REC) in a model of Wilson's disease[END_REF] In addition to biological investigations, molecular characterisation of the ATP7B gene can reveal disease-specific mutations or haplotypes. Most WD patients are mixed heterozygotes (carrying two different ATP7B gene mutations) and the relationship between the ATP7B genotype and the disease phenotype has proven difficult to decipher. Additional epigenetic, environmental and genetic factors (e.g. other genes involved in copper metabolism) are likely to strongly influence WD pathogenesis and clinical phenotype. [START_REF] Kieffer | Wilson disease: At the crossroads between genetics and epigenetics-A review of the evidence[END_REF][START_REF] Lv | Recent advance in the molecular genetics of Wilson disease and hereditary hemochromatosis[END_REF][START_REF] Medici | Genetic and environmental modifiers of Wilson disease[END_REF] To facilitate WD diagnosis and as part of a plan to screen new-borns, Jung and coworkers [START_REF] Jung | Quantification of ATP7B Protein in Dried Blood Spots by Peptide Immuno-SRM as a Potential Screen for Wilson's Disease[END_REF] recently proposed a targeted proteomic assay that directly quantifies ATP7B in dried blood spots (DBS). Promising results were obtained, with DBS from 12 WD patients showing reduced concentrations or lack of ATP7B. However, the authors warned that complementary biological tests remain necessary to diagnose patients carrying mutations which have a functional impact without affecting ATP7B expression levels. [START_REF] Jung | Quantification of ATP7B Protein in Dried Blood Spots by Peptide Immuno-SRM as a Potential Screen for Wilson's Disease[END_REF] In addition to the search for new diagnostic tests, attempts have been made to study the pathophysiology of WD, and thus to facilitate the development of new therapeutic compounds. For example, in 1999, Buiakova and coworkers [START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF] developed the Atp7b -/-mouse model. This model is now well-established as a genetic and phenotypic model for the hepatic form of WD. In this murine model, stop codons were introduced into exon 2 of the ATP7B gene, resulting in the production of a truncated mRNA and suppression of ATP7B expression in the liver. Like WD patients, Atp7b -/-mice accumulate copper in the liver, excrete copper in their urine and progressively develop liver disease. [START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF] Copper overload is also observed in other organs (such as kidney and brain) at later stages, although neurological symptoms are generally not observed in Atp7b -/-mice. Without chelation therapy, liver disease in Atp7b -/-mice progresses through the major successive stages. Liver disease is absent and no histological evidence of the disease is observed up to 8-12 weeks after birth, although underlying molecular changes alter the cell cycle machinery and lipid metabolism. [START_REF] Huster | High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease[END_REF][START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF] After 12 weeks, signs of hepatocellular injury are present, with hepatocyte necrosis and lobular inflammation. Bile duct proliferation is also observed alongside metabolic modifications. From 28 weeks of age, liver inflammation and fibrosis are observed alongside regenerating parenchyma and proliferating bile ducts. [START_REF] Lutsenko | Atp7b-/-mice as a model for studies of Wilson's disease[END_REF][START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF] In an attempt to gain new insights into the pathophysiological changes associated with WD and with a view to identifying new biomarkers to facilitate diagnosis, we investigated the plasma proteome modifications associated with liver disease development in the Atp7b -/-mouse model. As most plasma proteins are secreted by the liver, we hypothesised that the disruption of copper homeostasis and subsequent liver dysfunction in the Atp7b -/-mouse could significantly affect the plasma proteome. To characterise the quantitative modifications occurring in the plasma proteome in Atp7b -/-mice compared to wild-type (WT) Atp7b +/+ littermates, we first conducted a label-free quantitative (LFQ) mass spectrometry (MS)-based discovery proteomics study. To confirm the modifications detected, we developed a targeted quantitative proteomics assay using liquid chromatography-selected reaction monitoring (LC-SRM). This assay was applied to plasma samples from a distinct, larger, cohort of Atp7b -/-and WT mice. Using this strategy, we identified seven plasma proteins expressed at differential abundances between Atp7b -/-and WT mice. We then went on to determine their relevance to human disease, by investigating these proteins in clinical samples from WD patients, non-alcoholic steatohepatitis (NASH) patients, and healthy donors.

Experimental Mice and plasma samples

The Atp7b -/-mouse model used in this study was developed by Buiakova et al. [START_REF] Buiakova | Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation[END_REF] 129S6/SvEv Atp7b -/-males were crossed with C57BL/6J WT females before embryo transfer into recipient female mice. Atp7b +/-heterozygous mice (maintained on a mixed 129S6/SvEv x C57BL/6J genetic background) were bred in our animal care facility and used to generate Atp7b -/-and Atp7b +/+ mice. This breeding scheme prevents death of Atp7b -/- mice shortly after birth due to copper deficiency. Indeed, Atp7b - /-mice are born copper-deficient and delivery of copper from the mammary gland to the milk is impaired in Atp7b -/-mothers. Breeding, genotyping, and all experimental procedures were performed according to protocols approved by the ethics committees (C2EA -12 Comité d'éthique ComEth Grenoble and C2EA -44 CETEA -CEA DSV IdF), the veterinary authorities, and the French Ministry for Research. Blood samples were collected in sodium heparin-treated tubes (Becton Dickinson) by cardiac puncture from Atp7b -/-and WT mice (males and females) of various ages (Table 1). Blood samples were immediately stored at 4 °C. Within one hour of collection, they were centrifuged at 2,000 g and 4 °C for 15 min to collect plasma supernatants, which were aliquoted and immediately stored at -80 °C.

Patients and plasma samples

Plasma samples from 20 WD patients (13 women and 7 men, mean age: 41 ± 12 years) were collected at the French Reference Centre for Wilson's disease (Lyon and Paris, France). Plasma samples from 10 NASH patients (5 women and 5 men, mean age: 49 ± 8 years) and 10 healthy subjects with no history of chronic liver disease (5 women and 5 men, mean age 45 ± 13 years) were collected at the Clinique Universitaire d'Hépatogastroentérologie (Grenoble, France). The study protocols were approved by the ethics committee (Comité d'Evaluation Ethique de l'Inserm CEEI/IRB and Grenoble hospital's institutional review board). All patients gave written informed consent for their participation in this study. Plasma samples were collected by vein puncture and using lithium heparin-treated or EDTAtreated tubes (Becton Dickinson). Previous proteomics studies indicated that detection of the proteins targeted in this study is not influenced by anticoagulant [START_REF] Ilies | Data on the impact of the blood sample collection methods on blood protein profiling studies[END_REF][START_REF] Ilies | Impact of blood sample collection methods on blood protein profiling studies[END_REF][START_REF] Lan | Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics[END_REF] . Blood samples were stored at 4 °C for up to one hour before centrifugation at 2000 g and 4 °C for 15 min to isolate plasma. Plasma samples were anonymised, aliquoted and stored at -80 °C until use. Clinical data and biological parameters for the 30 recruited patients are presented in Supplemental Table 1.

Biochemical preparation of plasma samples

Plasma samples were prepared by a protocol adapted from the MED-FASP (multiple enzyme digestion -filter aided sample preparation) protocol. [START_REF] Wisniewski | Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis[END_REF] For each sample, 3 µL of plasma was loaded onto a 10 kDa cut-off ultrafiltration device (Amicon). Plasma proteins were denatured and reduced on the device by adding 4 M urea, 25 mM ammonium bicarbonate and 10 mM TCEP. Each sample was washed twice with 4 M urea, 50 mM ammonium bicarbonate and then alkylated with 55 mM iodoacetamide in 4 M urea, 25 mM ammonium bicarbonate. After two additional washing steps, the sample volume was reduced to 25 μL and proteins were digested for 3 h at 37 °C using trypsin/LysC mix (Promega) at a protein/enzyme ratio of 1:20 (w/w). The urea concentration was reduced to 1 M by dilution, and digestion was allowed to proceed for a further 3 h at 37 °C. Proteolytic peptides were recovered by adding 50 µL NaCl 0.5 M to the filter and centrifuging for 40 min at 14 000 g at room temperature. When performing targeted proteomics analyses, defined concentrations of isotope-labelled peptides (crude Pepotec grade, Thermo Fischer Scientific) were spiked into the digested samples at this stage of the protocol (see Table 2). All peptide digests were purified on Macrospin C18 columns (Harvard apparatus) in line with the manufacturer's recommendations before drying by vacuum centrifugation.

NanoLC-MS/MS analyses

Dried peptide digests were solubilised in 2 mL of 5% acetonitrile, 0.1% formic acid; 1 μL (equivalent to 100 ng protein) of this solution was analysed by nanoflow liquid chromatography (nanoLC) coupled online to MS (Ultimate 3000 and LTQ Orbitrap Velos Pro, Thermo Scientific). Peptides were sampled on a 300 µm x 5 mm PepMap C18 precolumn and separated on a 75 µm x 250 mm C18 column (Pepmap, Thermo Fischer). The nanoLC method consisted in a 120-min gradient at a flow rate of 300 nL/min, ranging from 5% to 37% acetonitrile in 0.1% formic acid for 114 min, followed by a ramp up to 72% acetonitrile in 0.1% formic acid for the last 6 min. MS and MS/MS data were acquired using Xcalibur (Thermo Fisher Scientific). The spray voltage was set to 1.4 kV, and the heated capillary was maintained at 200 °C. Survey full-scan MS spectra (m/z = 400-1600) were acquired in the Orbitrap at a resolution of 60 000 after the accumulation of 10 6 ions (maximum filling time: 500 ms). The 20 most intense ions from the preview survey scan delivered by the Orbitrap were fragmented by collision-induced dissociation (collision energy: 35%) in the linear trap (LTQ) after accumulation of 10 4 ions (maximum filling time: 100 ms).

Processing nanoLC-MS/MS data

Raw data were processed using MaxQuant software (version 1.5.8.3). Spectra were searched against the UniProt database (Mus musculus taxonomy) and the frequently observed contaminants database embedded in MaxQuant. Trypsin/LysC was selected as the enzyme, and up to two missed cleavages were allowed. Precursor mass error tolerances were set to 20 ppm and 4.5 ppm for initial and main searches, respectively. Peptide modifications allowed during the search were: carbamidomethylation (C, fixed), acetyl (Protein N-term, variable) and oxidation (M, variable). Minimum peptide length was set to seven amino acids. Minimum number of peptides, razor + unique peptides and unique peptides were all set to 1. Maximum false discovery rates (FDR) -calculated by employing a reverse database strategy -were set to 0.01 at both peptide and protein levels. Intensity-based absolute quantification (iBAQ) values were calculated from MS intensities of unique + razor peptides. The MS proteomics data have been submitted to the ProteomeXchange Consortium via the PRIDE partner repository [START_REF] Vizcaino | update of the PRIDE database and its related tools[END_REF] under dataset identifier PXD011007. Statistical analyses were performed using ProStaR. [START_REF] Wieczorek | DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics[END_REF] Proteins identified in the reverse and contaminant databases and proteins identified by a single peptide, as well as proteins for which fewer than 4 iBAQ values were available in a single condition, were removed from the list. After log2 transformation, iBAQ values were normalised by median centring before imputing missing values (missing values were replaced by the 2.5 percentile value for each sample); statistical testing was performed using a Limma moderated t-test. Differentially recovered proteins were sorted out using a log2 fold-change (FC) cut-off of 1.5 (between Atp7b - /-and WT mice samples) and a p-value threshold (on the remaining proteins) that guarantees a Benjamini-Hochberg FDR < 5%.

LC-SRM analyses

The peptides monitored during targeted analyses were selected based on: i) sequence-specificity by BLAST search against UniProt database (Mus musculus or Homo sapiens taxon), ii) analytical detectability predicted based on their ESPPredictor score, [START_REF] Fusaro | Prediction of high-responding peptides for targeted protein assays by mass spectrometry[END_REF] iii) absence of post-translational modifications, and iv) absence of reactive amino acid residues (C, M, N-terminal Q). SRM transition lists used to monitor the selected signature peptides were generated using Skyline. [START_REF] Maclean | Skyline: an open source document editor for creating and analyzing targeted proteomics experiments[END_REF] Labelled versions of the selected signature peptides (crude Pepotec grade) with Cterminal [ 13 C6, [START_REF] Ilies | Impact of blood sample collection methods on blood protein profiling studies[END_REF] N2]-lysine or C-terminal [ 13 C6, [START_REF] Ilies | Impact of blood sample collection methods on blood protein profiling studies[END_REF] N4]-arginine were purchased from Thermo Scientific. These isotopically labelled peptides were spiked into pre-digested plasma and analysed by LC-SRM to experimentally select the most responsive SRM transitions, optimise the LC gradient, and schedule acquisition. For optimal detection, two distinct LC-SRM acquisitions were performed when analysing murine plasma samples: (i) the first targeted alpha-fetoprotein, soluble transferrin receptor 1, carboxylesterase 2, complement C9, immunoglobulin J chain, and SERPINA3M; (ii) the second targeted clusterin and ceruloplasmin (Supplemental Table 1). LC-SRM analyses were performed on a 6500 QTrap hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex) with a TurboV electrospray ion source. Data were processed using Analyst software (version 1.6, AB Sciex). The mass spectrometer was linked to an Ultimate 3000 LC-chromatography system (Thermo Scientific). Chromatographic separation was achieved using a two-solvent system combining solvent A (2% acetonitrile, 0.1% formic acid) and solvent B (80% acetonitrile, 0.1% formic acid). Before separation, peptide digests were concentrated on a C18 precolumn (Phenomenex, ref: AJO-8782). Peptides were separated on a Kinetex C18 column (2.1 mm x 100 mm, Core-shell 2.6 µm, 100 Å, Phenomenex, ref: 00D-4462-AN) by applying a linear gradient from 4% to 14% B in 27 min, from 14% to 35% B in 8 min, and from 35% to 90% B in 4 min at a flow rate of 60 µL/min. MS data were acquired in positive mode with an ion-spray voltage of 4100 V; curtain gas was used at 45 p.s.a and the interface heater temperature was set to 250 °C. Collision cell exit, declustering and entrance potentials were set to 21, 55 and 14 V, respectively. Collision energy (CE) values were calculated using linear equations based on the unlabelled peptide precursor m/z ratios: CE = 0.044 m/z + 5 and CE = 0.05 m/z + 4 (Volts) for doubly-and triply-charged precursors, respectively, and the same CE was used for both labelled and unlabelled versions of each signature peptide. The analyses combined in the same run: (1) a precursor ion scan between 400 and 1000 m/z as a survey scan for Information-Dependent Acquisition (IDA), (2) an Enhanced Product Ion (EPI) scan with a scan speed of 1000 amu/sec and a dynamic fill-time for optimal MS/MS analysis, (3) an SRM acquisition with Q1 and Q3 quadrupoles operating at unit resolution. The acquisition time window for scheduled SRM analyses was set to 120 s, and the target scan time was set to 1.5 s. With a mean base width of 20 s, 13 points were acquired per LC peak. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [START_REF] Vizcaino | update of the PRIDE database and its related tools[END_REF] under dataset identifier PXD011007.

LC-SRM data processing

LC-SRM data were analysed using Skyline software. [START_REF] Maclean | Skyline: an open source document editor for creating and analyzing targeted proteomics experiments[END_REF] Peak picking was performed using the mProphet algorithm and the "second best peak" model. A Q-value of 0.01 (1% FDR) was set as the cut-off for peptide signal analysis. In addition to peptide signal scoring, all transitions were visually inspected and excluded if they were incompatible with quantification (low signal-to-noise ratio, obvious interference). Unlabelled/labelled peak area ratios were calculated for each SRM transition and were averaged to determine the corresponding peptide ratio. At least two transition pairs were used to determine biomarker concentrations. Protein ratios were calculated from the ratios obtained for signature peptides. Finally, candidate biomarker concentrations were calculated from the average protein ratio and the concentration of isotope-labelled peptide added to the sample, as estimated by the provider. Plasma protein concentrations determined using LC-SRM were compared between groups of mice. For each protein, statistical significance was evaluated on the basis of a FC > 2 or < -2 and a p-value < 0.05 (Mann-Whitney test). Significance of protein concentration differences in human plasma samples was analysed by one-way ANOVA, followed by the Tukey multiplecomparison post-hoc test. The threshold for significance was set to p < 0.05.

ELISA assays

Alpha-fetoprotein (FETA) and carboxylesterase 2 (CES2) concentrations in human plasma samples were determined using the alpha-fetoprotein ELISA kit (reference ab193765, Abcam) and the CES2 ELISA kit (reference ABIN420902, Cloudclone corp), respectively, according to the manufacturers' instructions.

Results

Discovery LFQ proteomics to explore the Atp7b -/-mouse plasma proteome

We initially characterised and compared the plasma proteomes of Atp7b -/-and WT mice using unbiased LFQ proteomics. For this discovery phase, plasma samples from 8 WT mice and 13 Atp7b - /-mice were collected from 36-and 44-week-old animals (Table 1). In this age range, Atp7b -/-mice had a 50-fold higher intrahepatic copper concentration than WT mice. Atp7b -/-mice also exhibited advanced liver disease with intense inflammation and fibrosis (as previously described [START_REF] Heissat | Evaluation of the accuracy of exchangeable copper and relative exchangeable copper (REC) in a model of Wilson's disease[END_REF] ). Plasma samples were prepared using an adapted MED-FASP protocol and were analysed by nanoLC-MS/MS (Fig. 1A). Data processing using two significant peptides per protein and an FDR of less than 1% at both peptide and protein levels resulted in the identification of 367 unique proteins in the 21 plasma samples (Supplemental Table 1).

To assess differences in plasma protein abundance between Atp7b -/-and WT mice, a Limma test (with Log2 FC > 1.5 or < -1.5 and p-value < 0.05) was applied to the LC-MS/MS dataset. Upon applying these parameters, 18 of the 367 unique proteins were identified as differentially abundant between the two groups of mice. Of these proteins, 15 were enriched in Atp7b -/-plasma samples, and the remaining three were more abundant in WT plasma samples (Fig. 1B, Table 2). Interestingly, plasma levels of ceruloplasmin were not found to differ between Atp7b -/-and WT mice.

Targeted quantitative proteomics to validate changes to the Atp7b -/-mouse plasma proteome

Following the discovery phase, a quantitative LC-SRM assay was developed to confirm the differential abundance of the selected proteins on a distinct of plasma samples collected from 24 Atp7b -/-and 24 WT mice aged between 12 and 56 weeks (Table 1). This range of ages covers all stages of liver disease progression in this model animal. The initial list of protein targets consisted of the proteins identified in the discovery phase, with the exception of beta-globin, which was excluded from the panel studied as it is affected by haemolysis. Two additional proteins were added to the list: (i) ceruloplasmin, which is a classical biological indicator used to diagnose WD in humans; and (ii) clusterin, which was included because it interacts with ATP7B [START_REF] Materia | Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B[END_REF] -although its FC in the discovery assay was 2.06 (below the cut-off), it had a very significant p-value (0.001; Table 2).

Table 2. Proteins assayed by targeted proteomics.

To develop the LC-SRM assay, the 19 target proteins were submitted to in silico digestion. Surrogate signature peptides were then selected for each protein based on sequencespecificity, absence of missed cleavage or post-translational modification, and analytical detectability. Then, isotopically labelled versions of the signature peptides were purchased, spiked into pre-digested plasma matrix and analysed by LC-SRM to allow us to select only the most responsive peptides and SRM transitions, and to schedule acquisition. Following these optimisations, four of the proteins selected based on the discovery phase results (proteasome subunit alpha type, fibulin-1, carboxypeptidase Q and cathepsin E) were found to be unquantifiable due to difficulties in finding specific and/or "flyer" peptides. Consequently, the final LC-SRM assay (based on two acquisition methods) included 15 target proteins, which were quantified based on monitoring of 31 signature peptides, in labelled and unlabelled forms (Supplemental Table 1). Plasma samples from 24 Atp7b -/-and 24 WT mice were prepared by the adapted MED-FASP protocol. All samples were spiked with labelled peptides in defined quantities before peptide purification (Fig. 2A). Following LC-SRM analysis and data processing, plasma protein concentration differences were investigated with respect to sex and genotype differences. Five of the 15 plasma proteins (haptoglobin, serine protease inhibitor A3M (SERPINA3M), carboxylesterase 2E, complement component C9, and clusterin) were differentially abundant between males and females (Supplemental Table 1). Seven of the 15 plasma proteins quantified were confirmed to be differentially abundant between samples from Atp7b -/-and WT mice (Fig. 3A). Plasma concentrations for carboxylesterase 2E, transferrin receptor isoform CRA (soluble form), complement component C9, immunoglobulin J chain, alpha-fetoprotein, and clusterin were significantly higher in Atp7b -/-mice. An agedependent increase mirroring disease progression in Atp7b -/- mice was clearly observed for transferrin receptor isoform CRA, alpha-fetoprotein, and clusterin (Fig. 3B, Supplemental Table 1). Plasma levels for SERPINA3M, a mouse orthologue of human alpha 1-anti chymotrypsin, were decreased in Atp7b -/-mice. In line with the discovery study results, plasma ceruloplasmin levels were similar in Atp7b -/-and WT mice (Fig. 3A).

Screening of biomarker candidates in WD patients

Following the identification of these seven differentially abundant plasma proteins in the Atp7b -/-mouse model, we wished to investigate whether these results were transferrable to human WD. Therefore, we modified our targeted proteomic assay to allow quantification of the seven human protein orthologues in plasma samples. Ceruloplasmin was once again included in the panel assessed, as its plasma concentration is classically decreased in WD and is used for diagnosis. As for the mouse study, signature peptides were selected for the eight target human proteins based on sequence-specificity, absence of modifiable amino acids or post-translational modifications, and analytical detectability. Labelled signature peptides were purchased and used to develop the assay (SRM transition selection, LC gradient optimisation and scheduled acquisition) (Fig. 4A, Supplemental Table 1). Following these optimisations, the "human version" of our LC-SRM assay was used to quantify six proteins (transferrin receptor protein 1, complement C9, immunoglobulin J chain, SERPINA3, clusterin and ceruloplasmin), which were monitored simultaneously based on levels of 14 signature peptides, present in both labelled and unlabelled versions. The transition list consisted of 80 SRM transitions (Fig. 4B). Signature peptides for the remaining two proteins, human carboxylesterase 2 and alphafetoprotein, were barely detectable in plasma matrix, but both proteins were readily quantified using complementary ELISA assays. Following these analytical optimisations, we determined whether significant differences in plasma protein concentration existed between WD patients and healthy donors. The 20 WD patients were recruited by the French Reference Centres for WD. These patients presented either liver disease or a combination of hepatic, neurological, and psychiatric symptoms. Importantly, at the time of plasma sample collection, all WD patients were being treated with copper chelators (D-penicillamine, triethylenetetramine) or zinc sulfate, and their liver function was thus restored, as determined clinically and biologically based on transaminase levels and prothrombin time values (Supplemental Table 1). To assess the specificity of protein abundance changes, the 8 plasma proteins were also measured in patients with another liver disease: NASH. NASH was selected as the control liver disease as it shares some major clinical and histological features with WD (steatosis, inflammation, fibrosis) [START_REF] Poujois | Challenges in the diagnosis of Wilson disease[END_REF] . The quantification results were analysed by one-way ANOVA analysis followed by a post-hoc Tukey test. The plasma concentrations of 5 proteins identified in the -/-mouse model were no different between the three groups of human subjects (Supplemental Table 1). However, the plasma concentrations of alpha 1-antichymotrypsin (SERPINA3) were significantly decreased in treated WD patients compared to healthy donors, but not different from levels measured in NASH patients. Plasma levels of carboxylesterase 2 were increased in NASH patients, but there was no significant difference between treated WD patients and healthy subjects. Finally, as expected in the human disease, plasma levels of ceruloplasmin were significantly decreased in WD patients compared to both healthy subjects and NASH patients (Fig. 5). In summary, plasma proteome changes in treated human WD were quite different to those observed in the Atp7b -/-mouse.

Discussion

In this study, we used a pipeline combining unbiased discovery and targeted quantitative proteomics to characterise the plasma proteome changes associated with disease development in the Atp7b -/-mouse, a well-established model of hepatic WD. The proteins for which changes were detected were then assayed in plasma samples from WD patients. To the best of our knowledge, this is the first study to comprehensively explore the plasma proteome of this preclinical model and to investigate the similarities and differences compared to the human disease. Seven proteins were identified in Atp7b -/-mice for which abundance in plasma was significantly altered (more than 2fold) compared to control littermates (Fig. 2C). Upregulation of alpha-fetoprotein, soluble transferrin receptor 1 and clusterin can clearly be linked to the pathogenic mechanisms of WD. Interestingly, plasma levels of alpha-fetoprotein were higher in Atp7b -/-than in WT mice, in line with the intense regeneration of liver parenchyma associated with disease progression. [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF] We also observed increased levels of soluble transferrin receptor 1 (sTFR1) in plasma from Atp7b -/-mice, suggesting plasma iron depletion. In line with this result, Merle and coworkers [START_REF] Merle | Evidence for a critical role of ceruloplasmin oxidase activity in iron metabolism of Wilson disease gene knockout mice[END_REF] reported altered serum iron parameters in Atp7b -/-mice due to reduced ceruloplasmin ferroxidase activity. This reduction in activity leads to default incorporation of plasma iron into apotransferrin and impedes iron efflux from the liver to the circulation. [START_REF] Hayashi | Compound overload of copper and iron in patients with Wilson's disease[END_REF] Our results indicated similar ceruloplasmin plasma levels between Atp7b -/-and WT littermates, which is consistent with the results of a previous study reporting normal serum ceruloplasmin levels in Atp7b -/-mice. [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF] In contrast, the same study reported a marked alteration in the balance between apoceruloplasmin (inactive) and holoceruloplasmin (copper-bound) in Atp7b -/-animals. [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF] We also detected a significant increase in plasma clusterin in Atp7b -/-mice. This protein has been directly linked to maintaining copper homeostasis, and is secreted by a large number of tissues including the liver. This oxidative-stressinduced protein acts as an intracellular and extracellular controller of protein homeostasis. [START_REF] Trougakos | The molecular chaperone apolipoprotein J/clusterin as a sensor of oxidative stress: implications in therapeutic approaches -a mini-review[END_REF] In the context of WD, Materia and coworkers [START_REF] Materia | Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B[END_REF][START_REF] Materia | Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B[END_REF] demonstrated specific molecular interactions between clusterin and ATP7B, and suggested a role for clusterin in quality control for and clearance of ATP7B. Our results also identified four additional deregulated proteins for which the precise mechanisms relating to WD pathophysiology have yet to be deciphered. Plasma carboxylesterase 2 (isoform e) was detected at higher levels in Atp7b -/-mice. Although not fully characterised, this isoform is a member of the CES2 hydrolase family, which is involved in clearing xenobiotics and regulating lipid metabolism in the murine and human liver. [START_REF] Li | Carboxylesterase 2 prevents liver steatosis by modulating lipolysis, endoplasmic reticulum stress, and lipogenesis and is regulated by hepatocyte nuclear factor 4 alpha in mice[END_REF] Soluble CES2 proteins are contained in the endoplasmic reticulum or secreted, they exhibit triacylglycerol and diacylglycerol hydrolase activity. This activity was shown to be strongly related to several metabolic diseases -including NASH -which share pathogenic and histological hallmarks with WD (liver steatosis, inflammation, and fibrosis). [START_REF] Ruby | Human Carboxylesterase 2 Reverses Obesity-Induced Diacylglycerol Accumulation and Glucose Intolerance[END_REF] The elevated concentration of Ces2e in Atp7b -/-mice may be linked to deregulated lipid metabolism, which has been described in this model. [START_REF] Huster | High copper selectively alters lipid metabolism and cell cycle machinery in the mouse model of Wilson disease[END_REF] Plasma levels of complement C9 and immunoglobulin J chain -both immune proteins -were also higher in Atp7b -/-mice. However, the precise molecular mechanisms which led to their increased plasma abundance are currently unknown. Finally, SERPINA3M, a murine orthologue of human alpha 1-anti chymotrypsin (AACT), [START_REF] Heit | Update of the human and mouse SERPIN gene superfamily[END_REF] is a typical acute phase protein, circulating levels of which dramatically increase in response to inflammation. Despite the hepatic inflammation present in Atp7b -/-mice, this protein was downregulated in their plasma compared to WT. During inflammation, AACT inhibits several serine proteases including cathepsin G. Cathepsin G is released at the site of inflammation, where it contributes to defence against pathogens, tissue remodelling, and inflammation. Excessive or prolonged cathepsin G activity, caused by insufficient serpin regulation, can result in tissue damage. [START_REF] Baker | SERPINA3 (aka alpha-1-antichymotrypsin)[END_REF][START_REF] Faber | The molecular basis of alpha 1antichymotrypsin deficiency in a heterozygote with liver and lung disease[END_REF] Accordingly, AACT deficiency has been linked to chronic liver disease and cirrhosis (in a similar manner to alpha-1 antitrypsin deficiency). [START_REF] Faber | The molecular basis of alpha 1antichymotrypsin deficiency in a heterozygote with liver and lung disease[END_REF][START_REF] Ortega | alpha(1)-Antichymotrypsin deficiency associated with liver cirrhosis[END_REF] Thus, the reduction of AACT levels in Atp7b -/-mice may contribute to (or modulate) hepatic injury during disease progression. Finally, to examine the translational value of the seven plasma proteins found to be deregulated in the Atp7b -/-mouse model, the French National Centres for WD recruited 20 patients. At the time of plasma collection, all WD patients were receiving a continuous treatment (penicillamine, triethylenetetramine or zinc sulfate) and their liver function had normalised. Ideally, we would have liked to include samples from drug-naïve patients (at the time of diagnosis), but given the low incidence of WD, no such samples were available within the time-frame for our study. Consequently, a limitation of this study is that protein changes related to copper overload and hepatic injury could not be explored using the available cohort. Nevertheless, the modifications directly triggered by the perturbed ATP7B function (such as the effects on ceruloplasmin plasma levels) could be investigated using these plasma samples. Following the targeted proteomics study of human plasma samples, it seems possible that the changes associated with murine disease progression may be switched off by chelator treatment in human WD patients. Indeed, only ceruloplasmin (the known biomarker) and alpha-1-antichymotrypsin were confirmed as deregulated in WD patients receiving treatment compared to healthy donors. Other potential reasons for the discrepancy between the human and mouse data may include dissimilar stages of the pathology development, at which samples were analyzed, and a significant regenerative capacity of mouse liver that is evident in older Atp7b -/-mice. These results are a clear demonstration of the importance of validating results obtained in animal models as they may not always directly translate to human disease. Our results confirmed the marked drop in ceruloplasmin plasma concentrations specifically in treated WD patients, in line with an impact of ATP7B dysfunction on accelerated ceruloplasmin clearance. Indeed, impairment of cooper incorporation into apo-ceruloplasmin results in the secretion of a protein that lacks any ferroxidase activity and is rapidly degraded in plasma. [START_REF] Hellman | Mechanisms of copper incorporation into human ceruloplasmin[END_REF] No such decrease was observed in the Atp7b -/-mouse model, although ceruloplasmin-mediated oxidase activity was previously shown to be markedly reduced in this model (due to the secretion of catalytically inactive apo-ceruloplasmin). [START_REF] Huster | Consequences of copper accumulation in the livers of the Atp7b-/-(Wilson disease gene) knockout mice[END_REF] This result suggests that the murine apo-ceruloplasmin is much more stable in plasma than the human protein. A more extensive investigation of alpha-1 antichymotrypsin in WD patients would be of interest. Plasma levels of this protein were significantly decreased in treated WD patients compared to healthy controls, and the difference between WD and NASH patients was close to the significance threshold (p-value = 0.09) in the small cohort available for this study. As copper deficiency has been associated with NASH [START_REF] Morrell | The role of insufficient copper in lipid synthesis and fatty-liver disease[END_REF] , these results suggest that alpha-1 antichymotrypsin plasma levels could be modulated by copper balance.

Conclusions

This study combined LFQ discovery proteomics and SRM-based targeted proteomics to identify plasma proteome modifications associated with disease development in an established model of hepatic WD, the Atp7b -/-mouse. Seven deregulated plasma proteins were identified by the efficient analytical workflow developed. These proteins provide new insights into the pathogenic mechanisms associated with disease progression in this mouse model. Plasma samples from drug-naïve WD patients could reveal further correlations, but for the moment further study should focus on one of these proteins, alpha-1antichymotrypsin, which showed a similar differential expression pattern in the treated WD patients available here.

Fig. 1

 1 Fig. 1 Discovery proteomics workflow allowed identification of 18 differentially-expressed proteins. A. Plasma sample preparation before discovery LFQ proteomics analysis. B. Volcano plot showing proteins with significantly different expression levels in plasma from Atp7b -/- compared to WT mice (orange points). The -log10 (adjusted p-value) was plotted against the log2 FC (Atp7b -/-/WT). The non-axial vertical lines indicate ±1.5 log2 FC; the non-axial horizontal line delimits p-value = 0.05 (5% FDR threshold).

Fig. 2 .

 2 Fig. 2. Validation of 15 selected proteins in murine plasma by targeted proteomics. A. Workflow for plasma sample preparation before targeted quantitative analysis. B. Extracted ion chromatogram for scheduled LC-SRM analysis of plasma samples. To improve readability, only 16 of the 31 peptides monitored have been assigned on the chromatogram.

Fig. 3

 3 Fig. 3 Seven proteins confirmed to be expressed at significantly different levels between Atp7b -/-and WT mice. A. Dot plots showing the concentrations of seven proteins confirmed to be differentially abundant between Atp7b -/-and WT mice, on the basis of a FC of 2 and a p-value < 0.05, (Mann-Whitney test). Quantification results obtained for ceruloplasmin are also presented. B. Age-related changes observed for alpha-fetoprotein, transferrin receptor isoform CRA, and clusterin in Atp7b -/-(filled symbols) and WT (open symbols) mice. Concentration values measured for male and female mice are distinguished by square and triangular symbols, respectively.

Fig. 4

 4 Fig. 4 Quantification of eight selected proteins in human plasma samples by targeted proteomics. A. Workflow for plasma sample preparation before targeted quantitative analysis. B. Extracted ion chromatogram for scheduled LC-SRM analysis of human plasma samples. To improve readability, only 10 of the 14 peptides monitored have been assigned on the chromatogram.

Fig. 5

 5 Fig. 5 Ceruloplasmin and alpha-1-antichymotrypsin expressed at

Table 1 . Groups and numbers of mice used for LFQ proteomics discovery analysis, and validation based on targeted proteomics.

 1

This study was supported by grants from the CEA, from the Fondation de la Chimie, from the "Investissement d'Avenir Infrastructures Nationales en Biologie et Santé" programme (ProFI project, ANR-10-INBS-08) and from the French National Research Agency (GRAL project, ANR-10-LABX-49-01).

Acknowledgements

We thank the team at EDyP for scientific discussions. We are grateful to Prof. Svetlana Lutsenko and Dr. Dominik Huster for kindly providing access to the Atp7b -/-mouse model, to Samuel Wieczoreck, Sandrine Miesch-Fremy and Khémary Um for technical support, to Fonds de Dotation Clinatec for fundraising support and to Maighread Gallagher-Gambarelli for editing services.

Conflicts of interest

There are no conflicts to declare.


Aneesh Vijayan

Laurye Van Maele

Delphine Fougeron

Delphine Cayet

Jean-Claude Sirard

The GM-CSF released by airway epithelial cells orchestrates the mucosal adjuvant activity of flagellin

The Toll-like receptor 5 (TLR5) agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive f a e activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellincontaining vaccines. Here, we looked at how the airway epithelial cells regulate the flagellindependent stimulation of antigen-specific CD4 + T cells and the antibody response in mice. Our results demonstrate that after sensing flagellin, airway epithelial cells trigger the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of antigen-specific CD4 + T cell count and antibody titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by airway epithelial cells orchestrates the cross-talk between cDC2s and CD4 + T cells and thus drives f a e adjuvant effect.

SINGLE-SENTENCE KEY POINTS

Flagellin induces GM-CSF production by airway epithelial cells GM-CSF transactivates lung conventional dendritic cells GM-CSF drives the mucosal adjuvant activity of flagellin/TLR5 signaling

Running title : Key role of epithelial GM-CSF for mucosal adjuvant

INTRODUCTION

Vaccines against infectious diseases is based on the administration of protective, immunodominant antigens that trigger antibody and T cell responses and thus result in the neutralization or death of the pathogenic microorganisms. Most antigens are formulated in a way that enables a rapid, intense, and long-lasting protective response. To optimize immunization, adjuvants can be included to vaccines in order to promote targeting and stimulation of the innate immune system both of which are essential for an appropriate vaccine response [START_REF] Del Giudice | Correlates of adjuvanticity: A review on adjuvants in licensed vaccines[END_REF].

Toll-like receptor (TLR) agonists trigger innate immune responses and are used as adjuvant of vaccination. There are many evidences that TLR signaling enhances vaccine efficacy by effectively priming the adaptive immune system [START_REF] Mata-Haro | The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4[END_REF][START_REF] Pulendran | The varieties of immunological experience: of pathogens, stress, and dendritic cells[END_REF][START_REF] Wille-Reece | Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates[END_REF][START_REF] Liang | Vaccine priming is restricted to draining lymph nodes and controlled by adjuvantmediated antigen uptake[END_REF]. In the context of vaccination, the cells that respond primarily to TLR agonists include professional antigen-presenting cells (APCs) such as dendritic cells (DCs), B lymphocytes or macrophages. However, many structural cells including epithelial cells ubiquitously express pattern recognition receptors (PRRs) -including TLRs and therefore respond to TLR agonists [START_REF] Whitsett | Respiratory epithelial cells orchestrate pulmonary innate immunity[END_REF][START_REF] Tan | TLR4 signaling in stromal cells is critical for the initiation of allergic Th2 responses to inhaled antigen[END_REF][START_REF] Zhang | Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by Toll-like receptor 5[END_REF][START_REF] Hammad | House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells[END_REF][START_REF] Sato | Induction of antiviral immunity requires Toll-like receptor signaling in both stromal and dendritic cell compartments[END_REF][START_REF] Price | A Map of Toll-like Receptor Expression in the Intestinal Epithelium Reveals Distinct Spatial, Cell Type-Specific, and Temporal Patterns[END_REF] . There is a large body of literature data on how monophosphoryl lipid A (an attenuated lipopolysaccharide derivative used in adjuvant formulation) influences structural and myeloid cells at the site of vaccine injection [START_REF] Mata-Haro | The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4[END_REF][START_REF] Van Maele | Toll-like receptor 4 signaling in hematopoietic-lineage cells contributes to the enhanced activity of the human vaccine adjuvant AS01[END_REF][START_REF] Didierlaurent | Enhancement of Adaptive Immunity by the Human Vaccine Adjuvant AS01 Depends on Activated Dendritic Cells[END_REF][START_REF] Didierlaurent | AS04, an aluminum salt-and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity[END_REF].

Indeed, monophosphoryl lipid A triggers TLR4 signaling in the structural cells, which in turn contributes to the acc e efficacy.

Bacterial flagellin is a TLR5 agonist and acts as a potent systemic and mucosal adjuvant [START_REF] Vijayan | Compartmentalized Antimicrobial Defenses in Response to Flagellin[END_REF].

A DC a e e e a f f a e ad a c , e ce a d e DC ac a strongly influenced by the immunization route. The adjuvant activity is driven either by the direct or indirect activation of APCs (especially DCs) [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Sierro | Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells[END_REF][START_REF] Means | The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells[END_REF][START_REF] Didierlaurent | Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response[END_REF][START_REF] Uematsu | Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5[END_REF][START_REF] Flores-Langarica | CD103(+)CD11b(+) mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin[END_REF][START_REF] Flores-Langarica | Intestinal CD103(+)CD11b(+) cDC2 Conventional Dendritic Cells Are Required for Primary CD4(+) T and B Cell Responses to Soluble Flagellin[END_REF]. For example, the long-term, T-celldependent antibody response against systemically administered flagellin from Salmonella (FliC) depends on TLR5 signaling in CD11b + CD103 + double-positive conventional DCs (cDCs) [START_REF] Flores-Langarica | CD103(+)CD11b(+) mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin[END_REF][START_REF] Flores-Langarica | Intestinal CD103(+)CD11b(+) cDC2 Conventional Dendritic Cells Are Required for Primary CD4(+) T and B Cell Responses to Soluble Flagellin[END_REF][START_REF] Kinnebrew | Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense[END_REF].

In contrast, the delivery of flagellin to the mucosae (especially the airways) potentiates first-line adaptive responses by structural cells (especially airway epithelial cells, AECs) [START_REF] Anas | Lung epithelial MyD88 drives early pulmonary clearance of Pseudomonas aeruginosa by a flagellin dependent mechanism[END_REF][START_REF] Janot | Radioresistant cells expressing TLR5 control the respiratory epithelium's innate immune responses to flagellin[END_REF][START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF][START_REF] Lee | Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9[END_REF][START_REF] Wilson | The Toll-like receptor 5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens[END_REF]. Thus, intranasal or intratracheal administration of flagellin induces immediate and transient TLR5 signaling in lungs [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Janot | Radioresistant cells expressing TLR5 control the respiratory epithelium's innate immune responses to flagellin[END_REF][START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF][START_REF] Munoz | Mucosal administration of flagellin protects mice from Streptococcus pneumoniae lung infection[END_REF][START_REF] Yu | Flagellin stimulates protective lung mucosal immunity: role of cathelicidin-related antimicrobial peptide[END_REF]. Notably, experiments bone-marrow chimera and microdissection of lung tissue showed that flagellin activates the structural epithelial airway cells but does not impact on the alveolar macrophages. TLR5-mediated activation of AECs, in turn activates lung DCs [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Lee | Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9[END_REF][START_REF] Wilson | The Toll-like receptor 5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens[END_REF][START_REF] Sharma | The TLR5 Agonist Flagellin Shapes Phenotypical and Functional Activation of Lung Mucosal Antigen Presenting Cells in Neonatal Mice[END_REF] However, the underlying mechanism remains poorly understood. Airway epithelial cells respond to flagellin by releasing a broad range of cytokines and chemokines (such as CCL20, IL-1 , or IL-1 ) that may impact lung DC maturation. It has been suggested that the AEC-derived cytokines IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) promote DC activation [START_REF] Lee | Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9[END_REF][START_REF] Wilson | The Toll-like receptor 5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens[END_REF]. Furthermore, AEC stimulation by flagellin enhances the influx of neutrophils and monocytes that may contribute as APCs. Recently, we showed that neither the IL-1 cytokine family nor neutrophils nor monocytes orchestrate f a e mucosal adjuvant activity [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF]. In experiments in the mouse (and in contrast to cholera toxin subunit B, a potent mucosal adjuvant), intranasally administered recombinant flagellin did not enter into the lung parenchyma and thus could not activate the DCs located there [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF]. We therefore sought to establish how cross-talk between AECs and lung cDCs promotes f a e mucosal adjuvant effect. To this end, we used an in vitro model based on (i) murine AECs grown at the air-liquid interface (ALI), and (ii) FMS-like tyrosine kinase 3 Ligand (FLT3L)-derived cDCs from murine bone marrow. We demonstrated that flagellin-stimulated AECs release GM-CSF in a TLR5dependent manner and thus enhance the frequency of activated type 2 CD11b + DCs (cDC2s).

Furthermore, activated cDCs loaded with an MHCII-restricted ovalbumin (OVA) peptide primed OVA-specific CD4 + T cell (OTII cells) and prompted rapid proliferation of the latter. Disruption of GM-CSF signaling abrogated the flagellin-and AEC-dependent activation of cDCs and the subsequent activation of OTII cells. We next tested the effect of flagellin-induced GM-CSF on lung cDCs in vivo. Epithelium-derived GM-CSF significantly increased the frequency of cDC2s in the lung-draining mediastinal lymph nodes (MdLNs). In mice, the antibody-based blockade of GM-CSF signaling reduced the number of activated cDC2 in the MdLNs. In turn, this drastically reduced titers of antigen-specific CD4 + T-cell-dependent IgG antibodies. Taken as a whole, our results suggest that AEC-derived GM-CSF initiates the cross-talk between lung CD11b + cDCs and antigen-specific T cells, and thus promotes the mucosal adjuvant effect of flagellin.

MATERIALS AND METHODS

Ethics statement. Animals were maintained in a pathogen-free facility in compliance with institutional guidelines at the Institut Pasteur de Lille (animal facility agreement #C59-350009).

All experiments complied with current national, institutional and European regulations and ethical guidelines, were approved by our Institutional Animal Care and Use Committee (reference:

APAFIS#5164, protocol 2015121722429127_v4) and were conducted by qualified, accredited personnel.

Animals

Six-to eight-week-old female C57BL/6JRj mice (Janvier Labs, Saint Berthevin, France), OT-II, Tlr4 -/-and Tlr5 -/-mice (backcrossed on C57BL/6J) were used.

Reagents

The custom-designed flagellin primarily used in the present study (recombinant flagellin FliC 174- 400 harboring a carboxy-terminal histidine Tag) derives from Salmonella enterica serovar Typhimurium FliC [START_REF] Nempont | Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity[END_REF][START_REF] Porte | A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections[END_REF]. The recombinant FliC 174-400/89-96* (referred to henceforth as FliCTLR5mut, and which does not signal through TLR5) was generated by replacing the residues involved in TLR5 detection (89-96, QRVRELAV) with the corresponding sequence from a nonsignaling flagellin (DTVKVKAT) [START_REF] Munoz | Mucosal administration of flagellin protects mice from Streptococcus pneumoniae lung infection[END_REF][START_REF] Nempont | Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity[END_REF][START_REF] Porte | A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections[END_REF]. The recombinant flagellins were expressed in inclusion bodies in Escherichia coli BL21(DE3) and then purified to homogeneity with metal affinity, ion-exchange, gel filtration and fast protein liquid chromatography techniques (GE Healthcare, Pittsburgh, PA) after endotoxin depletion. Using a Limulus assay (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Thermo Fisher Scientific, Waltham, MA), we estimated the final endotoxin content in the protein preparation to be 0.016 endotoxin units per µg of protein.

The molecular consistency and biological activity of FliC 174-400 and FliCTLR5mut were quality-controlled as described in Supplementary Figure 1 A-C. Endograde OVA (Hyglos, Bernried, Germany) and OVA-specific MHCII-restricted peptide (Invivogen, San Diego, CA) were used in the present study. The anti-GM-CSF antibody (clone MP1-2EE9) and the isotype control (clone 2A3) were obtained from BioXcell, West Lebanon, NH. Recombinant murine FMS-like tyrosine kinase 3 ligand (FLT3L) was purchased from Peprotech, Neuilly-Sur-Seine, France.

Immunization and sample collection

Mice were lightly anesthetized (using gaseous isoflurane) prior to intranasal immunization/treatment in a maximum volume of 30 l. Unless otherwise specified, a homologous prime-boost immunization protocol was used to assess the specific adaptive immune responses to OVA (10 µg) as the model antigen and the flagellin FliC 174-400 (2 µg) as the adjuvant. For cDC activation and time-course studies, mice were sacrificed at 6 hours and 18 hours following intranasal instillation of FliC (2 µg). For GM-CSF depletion studies, animals were treated with 60 µg of GM-CSF-specific antibody or isotype control 2 hours prior to immunization, with an additional 60 µg of antibody then administered intranasally at the same time as the flagellin and/or OVA. Bronchoalveolar lavage (BAL), MdLNs, lung, spleen and serum samples were collected as described previously [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF][START_REF] Nempont | Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity[END_REF]. Lung extract were prepared by homogenizing the lung in 2 ml of Tissue Protein Extraction Reagent (Pierce, Rockford, IL).

Antigen-specific immune responses.

Air-liquid interface (ALI) cultures of AECs, and the production of conditioned medium

Mouse AEC cultures were established as described previously [START_REF] You | Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population[END_REF][START_REF] Crotta | Type I and type III interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia[END_REF]. The AECs were isolated by overnight incubation of mouse trachea with 0.15% pronase (Roche) at 4ºC. The cells were seeded on collagen-coated Transwell ® inserts (Corning TM , Corning, NY; one insert per mouse).

The ALI cultures were e ab ed ce e ce a e e a e a ce e ceeded 1000 c 2 (usually within one week). The medium was then removed from the apical side. The AECs were treated by adding 10 µl of 1 µg/ml of flagellin FliC 174-400 or PBS alone (control) to the apical side of the insert. The conditioned medium was collected from the basal compartment after 24 h, aliquoted, and stored at -80ºC prior to the cytokine assay or to treatment of DCs.

Production of FLT3L-derived bone marrow DCs

Conventional DCs were derived from the bone marrow (BM) of animals, as described previously [START_REF] Naik | Cutting edge: generation of splenic CD8+ and CD8-dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures[END_REF][START_REF] Beshara | Alteration of Flt3-Ligand-dependent de novo generation of conventional dendritic cells during influenza infection contributes to respiratory bacterial superinfection[END_REF]. Briefly, BM cells were isolated from mice, and any red blood cells were lysed. Next, 10 7 cells were suspended in 5 ml of RPMI complete medium supplemented with 100 ng/ml of mouse recombinant FLT3L (Peprotech). The cells were seeded into a single well of a 6-well plate (Ultralow binding, Corning TM ). After 3 days of culture, the cell suspension was divided into two volumes, and 2.5 ml of fresh medium supplemented with 100 ng/ml of recombinant FLT3L was added to each. The process was repeated on day 6, and the cells were ready to use on day 9.

Flow cytometry

Cell suspensions of lung, MdLNs or spleen were obtained by enzymatic digestion with collagenase from Clostridium type IA (1 mg/mL, Sigma-Aldrich) and DNAse I (40 µg/ml, Sigma-Aldrich) for

Gene expression analysis

Total RNA from AECs was isolated with a Nucleospin RNA kit (Macherey-Nagel). The RNA was reverse-transcribed with a High-Capacity cDNA Archive Kit (Applied Biosystems), according to

Co-cultures of CD4 lymphocytes and DCs.

CD4 cells were isolated from the lymph nodes and spleen of OTII mice, using negative selection (CD4 isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were next stained CTV, acc d a fac e tructions. The conditioned media from flagellin-or PBS-treated AECs were incubated with 10 µg/ml of anti-GM-CSF or isotype antibody for 30 minutes at 37 C. FLT3L-derived BMDCs were treated with the antibody-pre-incubated, AECconditioned media for 18 hours. After washing with RPMI medium, the DCs were loaded with 2 µg/ml of MHCII-restricted, OVA-specific peptide (OVA323-339; Invivogen). Following a 4-hour incubation at 37ºC, the DCs were washed and then incubated with CTV-stained CD4 T cells at a ratio of 1:20 for 4 days. The proliferation of OT-II cells was assessed using flow cytometry.

Statistics

Statistical analysis was performed using GraphPad Prism version 8 software (San Diego, CA).

Groups were compared using a Mann-Whitney test (for two independent groups) or Kruskal-Wallis one-a ANOVA D -test (for three or more groups). Unless otherwise specified, the results were expressed as the mean ± standard error of the mean (SEM).

RESULTS

Flagelli ac i i a a ucosal adjuvant is dependent on TLR5 signaling and CD4 + T cells

To confirm the involvement of TLR5 signaling in flagellin mucosal adjuvant activity, mice were immunized intranasally on days 0 and 21 with ovalbumin alone or formulated with FliC 174-400 or FliCTLR5mut (i.e. the counterpart with a defect in TLR5 signaling). We found that mice immunized with OVA and FliCTLR5mut had very low serum OVA-specific antibody titers similar to mice immunized with OVA only (Figure 1A). In contrast, a combined formulation of OVA and FliC 174-400 was associated with 100-fold more elevated titers of serum OVA-specific antibodies.

The specific ex vivo stimulation of lung-draining MdLN cells with OVA enhanced CD4 + T cell but not CD8 + T proliferation solely in animals that had received FliC 174-400 and OVA (Figure 1B).

Concordantly, OVA-stimulated MdLN cells secreted high levels of IFN and IL-2 when animals were immunized with FliC 174-400 (Supplementary Figure 1D). However, this effect of immunization was not observed with cells isolated from spleen or lungs since no cytokines were detectable in supernatant of OVA-stimulated cells. Since the elevation in the antigen-specific CD4 + T cell count was correlated with the serum antibody titer, we further evaluated the contribution of CD4 + T cells to f a e c a ad a activity. Antibody-based depletion of CD4 + T lymphocytes prior to immunization drastically reduced the levels of OVA-specific antibodies (Figure 1C). Taken as a whole, these results demonstrated that FliC 174-400 mucosal adjuvant activity is TLR5-dependent and requires antigen-specific CD4 + T cells.

Airway epithelial cells sense flagellin via TLR5 to release GM-CSF

We and others have found that AECs are the primary targets for f a e c a activity [START_REF] Anas | Lung epithelial MyD88 drives early pulmonary clearance of Pseudomonas aeruginosa by a flagellin dependent mechanism[END_REF][START_REF] Janot | Radioresistant cells expressing TLR5 control the respiratory epithelium's innate immune responses to flagellin[END_REF][START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF][START_REF] Lee | Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9[END_REF][START_REF] Wilson | The Toll-like receptor 5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens[END_REF]. The AECs that make up a large proportion of lung structural cells release chemokines and cytokines, which in turn can recruit DCs, modulate the ce functions, and shape a potent immune response. We therefore sought to investigate the DC-modulating cytokines that are released by AECs in response to flagellin. To this end, we studied cultures of tracheal epithelial cells from in vitro ALI cultures. The cells differentiated into a complex set of AECs that recapitulated the cell composition and structures found in the bronchial epithelium. We found that administration of flagellin FliC 174-400 at the air interface stimulated the expression of several genes linked to innate immune signaling (Figure 2A). We observed that the mRNA expression of the Csf2gene (coding for GM-CSF) was significantly upregulated (~10 fold) 2 hours after flagellin treatment, whereas the genes for other DC-modulating cytokines (such as TSLP, IL-33 and IL-25) were not affected.

Moreover, the polarized secretion of GM-CSF into the basal compartment of flagellin-stimulated AECs was enhanced after 24 hours (Figure 2B). Next, we probed the contribution of TLR5 signaling on AECs to the production of GM-CSF, using AECs from Tlr5 -/-mice. We found that Tlr5 -/-AECs did not secrete any GM-CSF when stimulated with FliC 174-400 (Figure 2C).

Similarly, we demonstrated that freshly isolated alveolar epithelial cells (CD45 neg CD31 neg Ter119 neg Epcam + ) from naïve animals were stimulated by flagellin to upregulate Csf2 mRNA levels, whereas this did not occur in cells from Tlr5 -/-animals (Supplementary Figure 2A-B). Using intranasal flagellin instillation, we next investigated whether GM-CSF could be detected in the respiratory compartments. In agreement with our in vitro observations, GM-CSF was detected in the BAL (Figure 2D) of animals -most prominently 4 hours after the treatment. Interestingly, GM-CSF production was upregulated in mouse BAL after the administration of the native form flagellin FliC, whereas TSLP, IL-33 and IL-25 were barely detected (Supplementary Figure 2C-D). These data suggest that AECs detect flagellin and promote the secretion of GM-CSF in a TLR5-dependent manner.

Type 2 cDCs respond to GM-CSF released by flagellin-stimulated AECs

Since lung-resident cDC2 are required f f a e ad a effec , we next looked at how AECs interact with cDC2. To this end, total mouse BM cells were differentiated into cDCs using the growth factor FLT3L. The resulting cells closely resembled the DC population found in murine tissues, including cDC1s, cDC2s, and plasmacytoid DCs. To circumvent any direct activation of DCs by flagellin, we generated FLT3L-derived cDCs from Tlr5 -/-mice. The conditioned medium collected from the basal compartment of the flagellin FliC 174-400-treated AECs was incubated with FLT3L-derived cDCs and compared with mock-conditioned AEC medium (Figure 3A). Our initial experiments demonstrated that flagellin-conditioned medium was able to influence DCs.

This stimulation was characterized by an enhanced number of activated cDC2s, as demonstrated by the increased frequency of the CD80 + CD86 + -expressing CD11b + CD24 dim cDC2 population.

Since GM-CSF production was identified as being differentially regulated by FliC 174-400, we addressed e fac contribution to DC maturation by neutralizing it with a specific monoclonal antibody (Figure 3B-C). The neutralization of GM-CSF in flagellin-conditioned medium was associated with a significantly lower mean ± SD frequency of activated cDC2s (46.5±4.95%), relative to the control group of isotype-treated cells (72.4±6.26%). Moreover, the expression of the activation markers CD80 and CD86 was significantly higher when for cells exposed to flagellin-conditioned AEC medium and the isotype antibody, compared with mock-conditioned controls. This effect was completely blocked by GM-CSF-specific antibodies (Figure 3C). Taken as a whole, these data demonstrated that the GM-CSF produced by flagellin-stimulated AECs is essential for regulating the number and activation status of FLT3L-derived cDC2s.

GM-CSF improves the ability of cDCs to prime antigen-specific CD4 + T cells

To determine the contribution of GM-CSF-exposed cDC to the priming of CD4 + T cells, we carried out an in vitro experiment with conditioned-medium-treated, OVA-specific MHCII peptideloaded-DCs and CD4 + T lymphocytes from OTII mice (Figure 4A). Briefly, FLT3L-derived DCs that had been exposed to AEC-conditioned medium and antibodies were loaded with MHCIIrestricted OVA323-339 peptide prior to incubation with CTV-labeled OVA-specific CD4 + T cells from OT-II mice. By measuring the dilution of CTV, we evaluated the proportion of CD4 + T cells that had divided after exposure to antigen and DCs (Figure 4B). Mock-conditioned medium promoted the division of about 18.9% of the CD4 + T cells, whereas flagellin-conditioned medium significantly promoted the division of proliferating CD4 + T cells (25%) (Figure 4B-C). The neutralization of GM-CSF during the incubation of DCs with flagellin-conditioned medium strongly impaired T cell proliferation (Figure 4C). Remarkably, antibody treatment yielded proliferation levels similar to those obtained with mock-conditioned medium.

Given the influence of the DC-T cell interaction on T cell priming, we sorted cDC2s from the MdLN of mice that had been immunized with flagellin and OVA in the presence of a GM-CSFspecific antibody prior to incubation with CTV-stained OTII cells (Supplementary Figure 3A-C). Strikingly, cDC2s from anti-GM-CSF-treated mice were less efficient in priming the CD4 + T cells than cDC2s from mock mice. Therefore, our data suggest that the secretion of GM-CSF by flagellin-stimulated AECs orchestrates CD4 + T cell activation by promoting the functional maturation of cDC2s.

Flagellin-dependent GM-CSF production in the lung enhances the number of cDC2s in MdLNs

After being activated, lung cDCs migrate to the MdLNs, where they prime T cells and help to mount an effective adaptive response [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF][START_REF] Sharma | The TLR5 Agonist Flagellin Shapes Phenotypical and Functional Activation of Lung Mucosal Antigen Presenting Cells in Neonatal Mice[END_REF]. To validate our in vitro observations, we determined whether or not flagellin-mediated GM-CSF production in the lung drive the increase in the frequencies and activation status of MdLN DCs (Figure 5A). Using fluorescently labelled OVA (OVAAF647), we found that cDC2s are the predominant OVA-carrying DCs in the MdLNs when this protein is given intranasally at the same time as flagellin (Supplementary Figure 3D).

Intranasal instillation of flagellin increased the cDC2s (as a proportion of total DCs), compared with naïve animals (62% vs. 46%). In contrast, the proportion of cDC1s did not change (Figure 5B). Concomitantly, we observed a significantly lower proportion (51%) of cDC2s in animals that had received anti-GM-CSF antibody along with flagellin, relative to isotype-treated animals) (Figure 5C). Furthermore, one third of the animals that received anti-GM-CSF antibody had almost the same cDC2 levels as in naïve mice. Furthermore, the frequency of CD80 + CD86 + cDC2 was significantly lower in experiments with neutralized GM-CSF (Figure 5D). Lastly, the cDC2s were less activated, as evidenced by the significantly lower median fluorescence intensity for CD80 following GM-CSF-specific antibody treatment but not following treatment with an isotype control (Figure 5E). Taken as a whole, these data show that flagellindriven GM-CSF production in the lung significantly upregulates the proportion of activated cDC2s in lung-draining MdLNs.

In vivo neutralization of GM-CSF abrogates the mucosal adjuvant activity of flagellin

To demonstrate that modulating the function and number of cDCs in the MdLN can affect f a e mucosal adjuvant activity, we performed prime-boost immunization with flagellin and OVA antigen at the same time as the treatment with an anti-GM-CSF antibody or an isotype control (Figure 6A). After ex vivo stimulation with OVA, 1.53% of the MdLN CD4+ T cells isolated from animals immunized with flagellin and OVA and then treated with an isotype antibody were found to be dividing (Figure 6B). In contrast, the proportion of dividing CD4 + T cells in animals that received anti-GM-CSF was only 0.44%; this corresponded to a 3-fold relative reduction. To check whether the number of antigen-specific CD4 + T cells was correlated with the titer of antigenspecific antibodies, OVA-specific antibodies were assayed in the serum and BAL of immunized animals. Both titers were significantly lower in mice that received anti-GM-CSF antibody than isotype-treated mice (Figure 6C). Furthermore, we did not observe any correlation between the frequency of cDC1s and the titer of OVA-specific antibodies following GM-CSF neutralization (R 2 =0.21, p=0.0852) (Supplementary Figure 3E). However, we observed a strong, positive correlation between the frequency of cDC2s and the titer of OVA antibodies (R 2 =0.72, p<0.0001).

This correlation suggested that flagellin-dependent GM-CSF production in the lung is important for mediating f a e adjuvant activity via cDC2 activation.

DISCUSSION

Airway epithelial cells are known to secrete factors that can influence a wide range of immune cells in the lung [START_REF] Whitsett | Respiratory epithelial cells orchestrate pulmonary innate immunity[END_REF]. The AEC ability to drive immunological changes during allergen exposure has been well characterized. Our present data reveal a new role for AECs: promoting the mucosal adjuvant activity of flagellin by the local production of GM-CSF and transactivation of lung DCs.

Our in vitro ALI cultures of AECs and DCs provided a comprehensive view of how GM-CSF released from flagellin-stimulated AECs orchestrates the cross-talk between cDC2s and antigenspecific CD4 + T cells. This is consistent with in vivo data suggesting that GM-CSF recruits cDC2s into lung-draining lymph nodes and thus helps to prime antigen-specific CD4 + T cells. These results underline the importance of GM-CSF production by AECs f a e adjuvant activity.

GM-CSF is a pleiotropic cytokine expressed by both hematopoietic and non-hematopoietic cells, and is known to regulate the proliferation and differentiation of various hematopoietic cells, including DCs [START_REF] Hamilton | GM-CSF in inflammation[END_REF]. The release of GM-CSF by AECs and the critical role of the GM-CSF receptor in the development of DCs in nonlymphoid tissue suggest the existence of cross-talk between structural cells and hematopoietic cells [START_REF] Greter | GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells[END_REF][START_REF] Tazi | Evidence that granulocyte macrophage-colony-stimulating factor regulates the distribution and differentiated state of dendritic cells/Langerhans cells in human lung and lung cancers[END_REF][START_REF] Cates | Intranasal exposure of mice to house dust mite elicits allergic airway inflammation via a GM-CSF-mediated mechanism[END_REF][START_REF] Zhou | GM-CSF-licensed CD11b+ lung dendritic cells orchestrate Th2 immunity to Blomia tropicalis[END_REF][START_REF] Cao | Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response[END_REF]. The expression of Csf2 in flagellin-stimulated alveolar epithelial cells and in ALI AEC cultures was upregulated (Figure 2 and Supplementary Figure 2). This finding was further corroborated by the low levels of GM-CSF expression in flagellin-treated Tlr5 -/-AECs, and also fits with our previous report in which the flagellin-driven expression of Csf2 in murine lungs was restricted to radioresistant structural cells [START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF]. These results are in agreement with observations in which nasal epithelial cells upregulate the production of GM-CSF [START_REF] Cao | Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response[END_REF]. Further investigations using conditional knockout models where the Tlr5 and Csf2 genes are inactivated in the airway epithelial cell compartments will be instrumental to demonstrate unambiguously that the flagellin-mediated adjuvant activity is dependent on GM-CSF released by AECs.

Recent research has suggested that DC influence flagellin s mucosal activity when the adjuvant is delivered at the surface of airway mucosa lining either nasal or lung tissue [START_REF] Fougeron | Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract[END_REF][START_REF] Lee | Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9[END_REF][START_REF] Sharma | The TLR5 Agonist Flagellin Shapes Phenotypical and Functional Activation of Lung Mucosal Antigen Presenting Cells in Neonatal Mice[END_REF][START_REF] Cao | Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response[END_REF]. The functional activation of DC is not due to direct TLR5 signaling in DC (such as transepithelial DC)

but rather to activation relayed by TLR5-stimulated epithelium. Our study demonstrated that the adjuvant effect is primarily driven by cDC2s. It remains to be determined how GM-CSF specifically impacts cDC2. The cDC2 phenotype can vary from the CD11b + profile found in the lungs to the CD103 + CD11b + profile found in the gut mucosal compartment. By using FLT3Lderived BMDCs that are known to give rise to cDC2s and cDC1s (i.e. cells that closely resemble the cDCs found in the lung), we were able to identify the role of the cDC2s. Since flagellin can directly activate cDC2s (21-23) (due to their TLR5 expression), we used cDCs from Tlr5 -/-animals in order to analyze solely the flagellin-independent activation effect. It appears that native flagellin (and probably the recombinant flagellin FliC 174-400 used in the present study) is rapidly degraded in the airways and translocates poorly in the lung parenchyma [START_REF] Van Maele | Airway structural cells regulate TLR5-mediated mucosal adjuvant activity[END_REF]. Hence, the activation of cDC2s by flagellin in the lung mucosal compartment is dependent on factors that are released by AECs.

We demonstrated here that GM-CSF released by FliC 174-400-stimulated AECs can activate cDC2s

both in vitro and in vivo. Our in vitro data on FLT3L-derived cDCs stimulated with conditioned medium from FliC 174-400-activated AECs in the presence of GM-CSF-neutralizing antibodies suggest that GM-CSF is a critical or at least important cytokine in the process of cDC2 maturation (Figure 3). Next, GM-CSF neutralization studies in flagellin-treated mice concurred with the in vitro observations and evidenced a significantly lower number of activated cDC2s in the lungdraining lymph nodes (Figure 4). This finding is in line with literature reports on the importance of GM-CSF as a key mediator of DC-driven responses to aero-allergens [START_REF] Cates | Intranasal exposure of mice to house dust mite elicits allergic airway inflammation via a GM-CSF-mediated mechanism[END_REF][START_REF] Sheih | GM-CSF produced by the airway epithelium is required for sensitization to cockroach allergen[END_REF][START_REF] Willart | Interleukin-1alpha controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33[END_REF] but contrasts with previous reports in which flagellin-activated AECs promoted adaptive immunity through TSLP, IL-25 or IL-33 [START_REF] Lee | Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9[END_REF][START_REF] Wilson | The Toll-like receptor 5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens[END_REF]. This difference may be due to the various flagellins that were used, especially the native form that harbors the hypervariable domain, and display a stronger TLR5stimulating activity than the recombinant flagellin FliC 174-400 [START_REF] Nempont | Deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity[END_REF]. Consistently, a recent study highlighted that the hypervariable domain of flagellin contributes to the magnitude of TLR5

signaling [START_REF] Steimle | Flagellin hypervariable region determines symbiotic properties of commensal Escherichia coli strains[END_REF].

The current paradigm suggests that cDC2s in lung-draining lymph nodes are ideally placed to prime CD4 + T cells, whereas cDC1s mainly cross-present antigens to CD8 + T cells [START_REF] Anandasabapathy | Migratory CD11b(+) conventional dendritic cells induce T follicular helper cell-dependent antibody responses[END_REF]. We found a strong correlation between the number of cDC2s activated by AEC-derived GM-CSF on one hand and frequency of antigen-specific CD4 + T cells on the other. Co-culture experiments with CD4 + T cells and FLT3L-derived cDCs that had been exposed to the supernatant from flagellinstimulated AEC conditioned medium clearly showed the role of GM-CSF-licensed cDCs in driving CD4 + T cell proliferation (Figure 5). Moreover, the diminished antigen-specific CD4 + T cell response in flagellin-adjuvanted animals treated with GM-CSF antibodies clearly demonstrated the importance of cDCs in CD4 + T cell priming (Figure 6). These data correlate with observations on flagellin impact on B cell response and IgA production at the nasal mucosa [START_REF] Cao | Frontline Science: Nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway dendritic cells and subsequent IgA response[END_REF].

The primary efficacy readout for many vaccines is the elicitation of antigen-specific antibodies, which is usually dependent on CD4 + T cells. Our data suggest that intranasal administration of FliC 174-400 efficiently primes antigen-specific CD4 + T cells and elicits a strong mucosal and systemic antibody response (Figure 1). In response to flagellin-adjuvanted intranasal administration of OVA, a weak antigen-specific antibody response was observed in CD4-depleted animals and in animals treated with GM-CSF-neutralizing antibodies (Figure 6). One possible explanation for this would be a cascade effect of GM-CSF neutralization on cDC2s, which in turn downregulates antigen-specific CD4 + T cells and affecting overall antibody titers. It was recently reported that cDC2s are efficient vectors in the T follicular helper-dependent antibody response, due to their propensity to locate to the T-B cell border (48). Further work is needed to determine whether GM-CSF has a role in the precise anatomic localization of cDC2s within lymph nodes.

Overall, our data suggest that GM-CSF has a critical role in triggering lung cDC2s to drive f a e ac a a c a adjuvant. Determining how AECs and the GM-CSF they produce affect cDC lung function and activation will constitute an important step in the development of new respiratory adjuvants for vaccines against major pathogens. The biophysical characteristics of recombinant flagellin FliC 174-400 (expressed and purified from E. coli) was assessed as described in A-C. (A). Mass spectrometry analysis of the protein yielded a single peak of the expected molecular weight (29 kDa). The insert shows the SDS-PAGE analysis of the indicated amounts (in g) of recombinant protein, followed by Coomassie staining. (B) In vitro activity of the recombinant protein, as measured in a Caco-2 cell-based assay. The reporter cell line contains a luciferase transgene that is driven by the CCL20 promoter. Flagellin FliC 174-400 that lacked TLR5 activity (FliCTLR5mut) and trypsin-treated FliC 174-400 were used as controls. (C) In vivo functional analysis of FliC 174-400. Tlr5-/-, Tlr4-/-or C57Bl/6JRj (WT) mice were given an intranasal instillation of 2 g of FliC 174-400, PBS, or trypsin-treated FliC 174-400. BAL samples were taken at 6 hours, and the CCL20 level was determined using an ELISA. Cellular response to vaccination was analyzed as follows in (D): C57BL/6JRj mice (n=5) were immunized by intranasal instillation with OVA protein (10 µg) alone or OVA admixed with either FliC 174-400 or FliCTLR5mut flagellin (2 µg) in PBS as a prime-boost regimen administered on days 0 and 21. Mediastinal lymph nodes were sampled on day 28 and pooled, and OVA-specific cytokine production was assessed. Levels of IFN and IL-2 levels in the supernatant of OVA-stimulated MdLN cells were measured using an ELISA. The dotted line indicates the assay's limit of detection. The results were representative of two experiments (a pool of two MdLNs and a pool of three MdLNs per group).

  Ma e S d a-Curie grant agreement No. 657107 to AV.

  30 min at 37°C. For lungs, hematopoietic cells were recovered after centrifugation on Percoll 20% (GE Healthcare), incubated with anti-CD16/CD32 FcR blocking antibodies (clone 2.4G2), and then stained with specific antibodies. The following antibodies were used to study tissue cells and BMDC populations: anti-Gr1-FITC (clone RB6-8C5, BioLegend, San Diego, CA), anti-CD3 -FITC (clone 145-2C11, BD Biosciences), anti-B220-FITC (clone RA3-6B2; BioLegend), anti-CD64-PE (clone X54-5/7.1; BioLegend), anti-SiglecF-AF647 (clone E50-2440; BD Biosciences), anti-CD11b-BV786 (clone M1/70; BioLegend), anti-MHC II-PB (clone M5/114.15.2; BioLegend), anti-CD11c-PE/Cy7 (clone N418; BioLegend), anti-CD24-PerCP-Cy5.5 (clone M1/69; BioLegend), anti-CD45-APC-Cy7 (clone 30F11; BioLegend), anti-CD86-BV605 (clone GL1; BioLegend), anti-CD80-PE/Cy5 (clone 16-10A1; BioLegend). To study vaccine-induced OVA-specific T cells: anti-CD3 -FITC, anti-CD4-APC/Fire750 (clone RM4-5; BioLegend), anti-CD8-PeCy7 (clone 53-6.7; BioLegend), anti-CD45-BV650 (clone 30F11; BioLegend) and CellTrace TM Violet (CTV; Invitrogen) were used. Dead cells were excluded from the analysis by using Live/Dead Aqua dye (Invitrogen). Cells were stained with surface-specific antibodies for 30 min at 4ºC. Data were collected on a BD LSR Fortessa (BD Biosciences) and analyzed with FlowJo software (V10).

  QuantStudio 12K real-time PCR system (Applied Biosystems, Foster City, CA), using Power SYBR® Green PCR Master Mix (Applied Biosystems). Relative mRNA levels (2-C ) e e determined by comparing (i) the cycle thresholds (Ct) for the gene of interest and for Actb ( C ) a d ( ) e C a e f ea ed a d c ( C ). T e e b da f C a e to 35 cycles.

Figure 1 .

 1 Figure 1. Flagellin adj an ac i ie i e TLR5 signaling and CD4 T lymphocytes.

Figure 2 .

 2 Figure 2. TLR5 engagement on AECs promotes GM-CSF production.

Figure 3 .

 3 Figure 3. The in vitro activation of FLT3L-derived BMDCs by flagellin-stimulated AECs

Figure 4 .

 4 Figure 4. GM-CSF from AECs licenses DCs to stimulate antigen-specific CD4 lymphocytes.

Figure 5 .

 5 Figure 5. GM-CSF regulates the flagellin-mediated activation of DCs in MdLNs.

Figure 6 .Figure S1 .

 6S1 Figure 6. Neutralization of GM-CSF ed ce flagellin adj an ac i i .

Figure S2 .

 S2 Figure S2. Airway epithelium and conducting airways respond to flagellin stimulation (A-B)Freshly isolated lung alveolar epithelial cells are activated by flagellin. Lungs that were isolated from Tlr5-/-or C57BL/6JRj (WT) mice were digested with dispase to obtain a cell suspension. Following negative selection using magnetic beads and antibodies specific for CD45, CD31, and Ter119, the cells were analyzed for purity (A). The cells before and after selection were stained with an antibody specific for airway epithelial cells (Epcam+). To further differentiate between type I and type II alveolar epithelial cells, additional markers such as podoplanin (type I) and CD74 (type II) were included in the flow cytometry analysis (B). The alveolar epithelial cells were allowed to rest for 18 hours after isolation. Next, the cells were counted and seeded at 2 × 105 in each well of a 96 well and stimulated with 1 g/ml of FliCΔ174-400 in PBS for 6 hours. The RNA was isolated from stimulated cells and analyzed for the upregulation of the indicated genes. (C-D) Expression of potential DC-activating cytokines in mouse lung and broncho-alveolar lavages in response to intranasal administration of native flagellin. C57BL/6JRj mice (n=4 per group) were intranasally administered with native flagellin FliC (1 µg in 20 µl PBS). Total lung and broncho-alveolar lavages were sampled at 2, 4, 8, 18, 24, and 36 hours post-instillation. Lung protein extracts (C) and broncho-alveolar lavages (D) were prepared and cytokines were then measured by specific ELISA. The dotted line indicates the detection limit of the ELISA assay Data represent the mean ± SEM and are representative of 1-2 experiments (*p<0.05).

Figure S3 .

 S3 Figure S3. GM-CSF regulates the flagellin-mediated adjuvant effect by modulation of type 2 conventional dendritic cells (A-C) GM-CSF regulates the flagellin-mediated DC-dependent priming of CD4 lymphocytes. (A) Experimental design. C57BL/6JRj mice (n=5) were treated with a GM-CSF-neutralizing antibody or PBS two hours prior to and then upon administration of flagellin FliC 174-400. All treatments were given intranasally in a total of volume of 30 µl per treatment (OVA protein (10 µg) alone or with OVA and flagellin FliC 174-400 (2 µg)). At 18 hours, MdLN were sampled, and cDC2 were sorted as CD11c+MHCII+CD11b+CD103neg cells within the live CD45+LinnegSiglecFneg population. Then cells were co-cultured for 4 days with CTV-stained, naïve, OVA-specific CD4 lymphocytes from OTII mice. (B) Proliferation of CD4 T cells. The percentage of dividing CD4 T cells (as measured by the dilution of CTV fluorescence into live CD45+CD3+CD4+V 2+ cells) was measured using flow cytometry. (C) Representative histograms of CTV dilution. Groups were compared using a Mann-Whitney unpaired t test. (D)The flagellin-activated cDC2s that capture antigen following intranasal instillation are found in the MdLNs. C57BL/6JRj mice (n=8) were intranasally administered with 10 g of OVAAF647 or 2 g of FliC 174-400 in PBS. T e ce MdLNs were sampled 18 hours after immunization and stained for cDC1s and cDC2s. Ratios of OVAAF647-associated cDC1s and cDC2s to total cDCs in the MdLNs. Groups were compared in a one-a ANOVA T e -test. The results were representative of two independent experiments. (E). Neutralization of GM-CSF is correlated with the adjuvant effect of flagellin. The correlation between cDC1 or cDC2 counts and the titer of OVA-specific antibodies elicited by the immunization with FliC 174-400 and OVA along with GM-CSF-neutralizing antibodies. The results from OVA-specific antibodies that were elicited in animals after a prime-boost with FliC 174-400 and GM-CSF antibody were plotted against the total frequencies of cDC2s and cDC1s (gated on CD45 live cells) in the MdLNs (18 hours) after exposure to FliC 174-400 and GM-CSF antibody. Correlations were assessed by calculating Pearson coefficient.

ACKNOWLEDGMENTS

We thank Laurence Mulard and Sylvie Bay at Institut Pasteur, Paris for the mass spectometry analysis.

DATA AND MATERIALS AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS

AV performed all animal, RT-qPCR, ELISA, and flow cytometry experiments. DC prepared the DC cultures and performed the experiments on the human airway epithelial cells. DF and LVM performed the experiments with the native flagellin FliC. AV and JCS designed experiments and wrote the manuscript. JCS supervised the experimental work as a whole.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. specific IgG were measured using an ELISA. Groups were compared using a Mann-Whitney unpaired t test.

OVA-specific

Tslp