domain: chim sdv phys

text:
Laëtitia Chézeau

Lori A Kohlstaedt

Alain Le Faou

Frédéric Cosnier

Bertrand Rihn

Laurent Gaté
email: laurent.gate@inrs.fr

Proteomic analysis of bronchoalveolar lavage fluid in rat exposed to TiO2 nanostructured aerosol by inhalation

Keywords: titanium dioxide, lung inflammation, protein expression profile, short-term response, longterm response, NETosis

The pulmonary toxicological properties of inhaled titanium dioxide were studied using bronchoalveolar lavage fluid (BALF) cytology and proteomics analyses. Fischer 344 rats were exposed to 10 mg/m 3 of TiO2 nanostructured aerosol by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. As previously described, cytological analyses of BALF showed a strong inflammatory response up to 3 post-exposure days, which persisted however, at a lower intensity up to 180 days. In addition, using Multidimensional Protein Identification Technology (MudPIT), we identified a total of 107, 50 and 45 proteins (UniprotKB identifiers) differentially expressed in exposed rats immediately, 3 and 180 days after the end of exposure respectively. Increased levels of inflammatory proteins, members of proteasome, various histones, proteins involved in cytoskeleton organization, were noticed up to 3 days (short-term response). Some of these proteins were linked with Neutrophil Extracellular Trap formation (NETosis). Long-term response was also characterized by a persistent altered expression of proteins up to 180 days. Altogether, these results suggest that exposure to low toxicity low solubility nanomaterials such as TiO2 may induce long-term changes in the pulmonary protein expression pattern of which the physio-pathological consequences are unknown.

Introduction

Due to the growing use of nanomaterials in various industrial processes, the number of workers potentially exposed is increasing even though the toxicological properties of these chemicals are still not completely known. Since nanoparticles (NPs) may get aerosolized, inhalation represents the main route of occupational exposure. Then, inhalation studies with nanomaterials in animal models appear to be the most relevant approach to assess the hazards associated with exposure to such nanostructured aerosols. Titanium dioxide (TiO2) is one of the most widely produced and used nanomaterials worldwide. TiO2 NPs are used in coatings, paints, self-cleaning windows, food and healthcare products, pharmaceuticals, and cosmetics including sunscreens [START_REF] Shi | Titanium dioxide nanoparticles: a review of current toxicological data[END_REF].

Several studies have been published on the pulmonary toxicity of inhaled TiO2 NPs, mainly focusing on the inflammatory response and histopathological changes [START_REF] Shi | Titanium dioxide nanoparticles: a review of current toxicological data[END_REF][START_REF] Bermudez | Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles[END_REF]. However, such conventional toxicological approaches are not sufficient to identify the underlying molecular mechanisms of NP toxicity. Thus, complementary strategies such as toxicogenomics and proteomics studies are required [START_REF] Halappanavar | Transcriptional profiling identifies physicochemical properties of nanomaterials that are determinants of the in vivo pulmonary response[END_REF][START_REF] Sturla | Systems toxicology: from basic research to risk assessment[END_REF]. This is of high interest to assess the complex interplay between nanomaterials and living organisms as well as to identify any long-term adverse outcomes. Most studies involving classical toxicological assays and high content screening methods are limited to short post-exposure periods up to 30 days [START_REF] Halappanavar | Transcriptional profiling identifies physicochemical properties of nanomaterials that are determinants of the in vivo pulmonary response[END_REF][START_REF] Husain | Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation[END_REF][START_REF] Nikota | Meta-analysis of transcriptomic responses as a means to identify pulmonary disease outcomes for engineered nanomaterials[END_REF]. Only few studies have been published on longer post-exposure periods [START_REF] Rahman | Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses[END_REF][START_REF] Husain | Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice[END_REF].

In a previous work, we have shown using a transcriptomics approach, that following rat exposure by nose-only inhalation to a TiO2 nanostructured aerosol (NSA), 6 hours/day, 5 days/week for 4 weeks, many genes were differentially expressed in the lung of exposed animals up to 6 months after the end of animal exposure; without any significant histopathological changes except a strong inflammatory response that decreased overtime [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF].

In this study, we pursue our investigation of the pulmonary toxicological properties of this TiO2

NSA by looking at the changes of protein levels in the BALF of exposed animals. For this, biological samples were analysed immediately, 3 and 180 days after the end of exposure, to investigate shortand long-term effects.

Materials and methods

Aerosol generation and characterization

TiO2 P25 (Aeroxide ® P25, Evonik ® ) was purchased from Sigma Aldrich (Saint-Quentin Fallavier, France), and was a mixed-phase nanocrystalline powder composed of 80% anatase and 20% rutile crystallites, with an average primary particle diameter of 21.5 ± 7.2 nm. The physical and chemical characteristics of this nanomaterial have been previously published [START_REF] Cosnier | Design and Characterization of an Inhalation System to Expose Rodents to Nanoaerosols[END_REF]. Its main characteristics are presented in Table 1. TiO2 P25 aerosol was generated at a target concentration of 10 mg/m 3 . Taking into account the interspecies differences in terms of respiratory parameters and lung deposited dose, this concentration and exposure time relate to the 8 h weighted average occupational exposure of a worker at 0.3 mg/m 3 (the NIOSH recommended exposure limit for ultrafine TiO2 [START_REF]Occupational Exposure to Titanium Dioxide Current Intelligence[END_REF]); throughout his entire career.

The aerosol was produced using a rotating brush (RBG1000, Palas, Karlsruhe, Germany). Dry, filtered compressed air was used as the carrier gas. The aerosol was delivered simultaneously to animals maintained in nose-only restraining tubes placed in four 9-port-inhalation chambers (EMMS, Electro-Medical Measurement System, Bordon, UK). Simultaneously, control rats were exposed to filtered air using the same exposure setup [START_REF] Cosnier | Design and Characterization of an Inhalation System to Expose Rodents to Nanoaerosols[END_REF]. The aerosol and filtered air were conditioned at a temperature of 22 ± 2°C and a relative humidity of 55 ± 10%, in line with the OECD TG 412 guideline [START_REF] Oecd | OECD Guidelines for the Testing of Chemicals[END_REF].

Animal exposure

Animal experiments were performed according to the European and French regulations relating to the protection of animals used for scientific purposes (European Directive 2010/63/EU). Procedures were approved by the local Ethics Committee (C2EA-66) and the French Ministry for Research and Higher Education (authorization No. 00692.01). Thirteen-week-old male Fisher F344 rats (Charles River Laboratories, France) were housed in standard environmental conditions (relative humidity: 55 ± 10%; temperature: 22 ± 2°C and 12/12 h light/dark cycle). Water and a standard laboratory animal diet (A04, Safe diet) were freely available. Two weeks before exposure to TiO2 NSA, rats were gradually acclimatized to the restraining tubes. Animals were then exposed either to filtered air or TiO2 NSA at a concentration of 10 mg/m 3 for 6 h/day, 5 days/week for 4 weeks.

Tissue sampling, bronchoalveolar lavage

Immediately after the last 6-hour exposure, and then 3, and 180 post-exposure days (d0, d3 and d180 respectively), 5 animals per group were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg) and exsanguinated through the abdominal aorta. The trachea was cut and the lung was removed. Bronchoalveolar lavage (BAL) was performed on the left lung by introducing icecold phosphate buffered saline (PBS) and collecting it in conical tubes. Tubes were centrifuged for 5 min at 400 x g and 4°C. The supernatants were taken for mass spectrometry analyses and proteome profiler experiment. The cell pellets were resuspended in ice-cold PBS for cell counting and cytospin preparation. BAL cells were counted using a Cellometer Vision (Nexcelom Bioscience, Lawrence, MA) following acridine orange/propidium iodide staining. BAL differential cell counts were performed on cytospin preparations after May-Grünwald Giemsa staining.

Sample preparation and MudPIT analysis of BALF

BALF (n = 5 per control and exposed groups on d0, d3, and d180) was digested with trypsin. The peptides obtained were desalted and dried before loading on a column filled with 10 cm of Polaris C18 5μm packing material (Hichrom), and 4 cm of Partisphere 5 SCX (Whatman). The column was directly connected to an electrospray ionization source mounted on an LTQ linear ion trap mass spectrometer (Thermo-Fisher). Chromatographic separation of peptides was achieved at a flow rate of 300 nL/min delivered by an Agilent 1200 HPLC equipped with a split line. Peptides were eluted using a 10-step MudPIT procedure [START_REF] Fuchs | Proteomic analysis of ribosomes: translational control of mRNA populations by glycogen synthase GYS1[END_REF]. Data were analyzed using Integrated Proteomics Pipeline (IP2) with ProLuCID program [START_REF] Xu | ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity[END_REF]. The rat database used for identifications was downloaded from EMSEMBL on May 8, 2014. A database of common contaminants was added to the rat database and the whole was concatenated with a decoy database in which the sequence for each entry in the original database was reversed to provide for the calculation of false positives [START_REF] Peng | Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome[END_REF]. Peptide identifications were set to require fully tryptic ends with a false discovery rate of 0.005 at the peptide level. Proteins were included in the dataset if there was at least one peptide match better than a false discovery rate of 0.001. The final calculated false discovery rate averaged about 1% at the protein level over all datasets used. Normalization and relative quantitation were done using the spectral counting method that provides an easy way of identifying proteins with differing abundances between complex mixtures using shotgun proteomics data. These approaches used for quantitative proteomics analyses, are based on different spectral counting metrics. In this study, we used the normalized spectral abundance factor (NSAF) and the exponentially modified protein abundance index (emPAI) [START_REF] Mcilwain | Estimating relative abundances of proteins from shotgun proteomics data[END_REF][START_REF] Zybailov | Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae[END_REF]. Differentially expressed proteins between control and exposed group, were identified at each time-point, using an ANOVA (p < 0.05), available in the Identification Stat Compare program in IP2. Samples were statistically compared based on spectral counts. Thus, we obtained a list of differentially expressed proteins described in terms of ENSEMBL protein identifiers (ENSRNOP), at different post-exposure times. Then, UniProt Knowledge Base (UniProt KB) was used to convert ENSEMBL identifiers lists to Uniprot identifiers lists. These lists of differentially expressed proteins were analyzed using GeneCards® database and the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 database (https://david.ncifcrf.gov/). DAVID database was used to identify biological functions altered following exposure to TiO2 NSA. Proteins that share similar functions were clustered based on biological processes, cellular components and molecular functions. In this study, we mainly focused on significantly enriched Gene Ontology Biological Processes (GO BP) (significant enrichment was identified with a modified Fisher's exact test (EASE score); p < 0.05), or on GO BP belonging to annotation clusters with an Enrichment Score (Z score) greater than 1.3 [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF].

We also examined significantly enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) biological pathways (p < 0.05), a collection of manually-drawn pathway maps representing knowledge on molecular interactions and reaction networks.

Protein-protein interactions (PPI) of differentially expressed proteins in BALF, were investigated on d0, d3 and d180, using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database of physical and functional interactions v10.5 [START_REF] Szklarczyk | STRING v10: protein-protein interaction networks, integrated over the tree of life[END_REF]. Network nodes represent proteins and edges represent protein-protein associations. Line color of network edges indicates the type of interaction evidence. Thus, known interactions are represented in light blue line (this color indicates the presence of database evidence) and purple line (experimental evidence); predicted interactions in green line (neighborhood evidence), red line (fusion evidence) and blue line (co-occurrence evidence); and others interactions in yellow line (text mining evidence), black line (co-expression evidence) and violet line (protein homology). The analysis section gives some brief statistics of the inferred network, such as the number of nodes and edges. The average node degree is a number of interactions (at the score threshold) that a protein has on the average in the network. The clustering coefficient is a measure of how connected the nodes in the network are. Highly connected networks have high values.

The expected number of edges gives how many edges are to be expected if the nodes were to be selected at random. A small PPI enrichment p-value indicates that the nodes are not random and that the observed number of edges is significant.

Cytokine expression in BALF

BAL supernatants collected on d0, d3 and d180 were analyzed using the Proteome Profiler™ Rat Cytokine Array Kit, Panel A (R&D Systems, Minneapolis, MN) to determine released cytokine levels.

Briefly, supernatants were incubated with a cocktail of biotinylated detection antibodies before applying the mixture onto nitrocellulose membranes spotted with immobilized antibodies specific for 29 selected rat cytokines and chemokines: CINC-1 (Cxcl1), CINC-2α/β (Cxcl3), CINC-3 (Cxcl2), CNTF, Fractalkine (Cx3cl1), GM-CSF, sICAM-1, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-6, IL-10, IL-13, IL-17, IP-10 (Cxcl10), LIX (Cxcl5), L-Selectin, MIG (Cxcl9), MIP-1α (Ccl3), MIP-3α (Ccl20), RANTES (Ccl5), Thymus chemokine (Cxcl7), TIMP-1, TNF-α and VEGF. Bound proteins were detected with streptavidin-conjugated horseradish peroxidase (HRP). Membranes were washed and developed using the Chemi Reagent Mix TM ; then read using a ChemiDoc TM Imager (Bio-Rad).

Statistical analysis

Statistical analysis of BALF cytological data was performed using a two-way ANOVA (both determinants being treatment and time). Data from the Proteome Profiler experiment were analyzed using a Student's t-test. Changes were considered statistically significant at p < 0.05.

Results

BAL cytology and lung histopathology

BAL cytology results have been extensively described previously [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF]. Briefly, exposure to TiO2 aerosol triggered a strong increase of neutrophilic granulocytes up to d3 (0.01 ± 0.005 x 10 6 vs. 1.06 ± 0.24 x 10 6 cells/left lung on d0 and 0.03 ± 0.01 x 10 6 vs. 1.07 ± 0.4 x 10 6 cells/left lung on d3 in control vs. exposed groups respectively). This influx decreased drastically but remained significant (p < 0.05) on d180 (26.2 ± 15.9 x 10 3 vs. 99.4 ± 26.8 x 10 3 cells/left lung). Lymphocytes numbers were similar throughout the time course in control and exposed animals (data not shown). In addition, the number of alveolar macrophages was only increased in samples collected on d3 (0.85 ± 0.08 x10 6 vs.

1.45 ± 0.41 x10 6 in control vs. exposed groups respectively). Histopathological analysis [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF] showed that on d0 and d3, particle-laden macrophages were detected in the alveolar lumen from exposed animals. Moreover, proliferation of type II pneumocytes resulting in minimal epithelialization in alveolar walls and foci of granulomatous inflammation were observed in the alveoli of some exposed animals. On d180, in exposed animals, most of the particle-laden macrophages present in the alveolar lumen were limited to a few large aggregates, particularly next to the alveolar ducts and the terminal bronchioles with a minimal magnitude and a multifocal distribution. Alveolar wall surrounding these aggregates showed minimal epithelialization.

MudPIT analysis

As previously shown [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF], total proteins in BALF were significantly increased in exposed animals on d0 and d3 (0.17 ± 0.05 vs. 0.28 ± 0.06 mg/mL on d0 and 0.12 ± 0.04 vs. 0.25 ± 0.04 mg/mL in controls and exposed animals respectively). These protein amounts were similar on d180 (0.20 ± 0.06 vs. 0.20 ± 0.03 mg/mL in controls and exposed animals respectively).

A chromatography-based proteomics technique named MudPIT, was performed on BALF. In BALF samples from controls and TiO2-exposed animals, 1848, 2237 and 1726 proteins were identified on d0, d3 and d180 respectively. Statistical analysis of data revealed that among them 137, 78 and 51 ENSRNOP identifiers (Ensembl protein identifiers for Rattus norvegicus) were significantly differentially expressed (p < 0.05) on d0, d3 and d180, respectively. On day 0, 116 out of 137 ENSRNOP identifiers were successfully mapped to 107 UniprotKB identifiers (Uniprot Knowledge Base) (Supplementary Table 1). On day 3, 58 out of 78 ENSRNOP identifiers were successfully mapped to 50 UniprotKB identifiers (Supplementary Table 2). On day 180, 45 out of 51 ENSRNOP identifiers were successfully mapped to [START_REF] Liz | Silver nanoparticles rapidly induce atypical human neutrophil cell death by a process involving inflammatory caspases and reactive oxygen species and induce neutrophil extracellular traps release upon cell adhesion[END_REF] UniprotKB identifiers (Supplementary Table 3).

Functional analysis based on GO terms was performed using DAVID V6.7. Main common enriched GO BPs altered in BALF were related to proteolysis, inflammation, protein maturation, response to organic/inorganic substance, response to hormone stimulus, and cytoskeleton organization (Fig. 1). The levels of proteins involved in inflammatory response mainly increased in the short-term response phase. Among these proteins we found C4a, C4b, C4bpa, S100 Calcium Binding Protein A8 (S100a8), and Peroxiredoxin2 (Prdx2) (Supplementary Table 1 & 2). On d180, proteins associated with inflammation such as Kininogen 1 (Kng1), Coagulation Factor II and XII (F2, F12), C8b, and Serpin family A member 3 (Serpina3n) were mainly decreased (Supplementary Table 3).

Concerning proteins involved in proteolysis, a process known to be activated during inflammation, Cathepsin S and D (Ctss, Ctsd), members of the proteasome complex such as Psma3, Psma4; were overexpressed in the short-term response phase (Supplementary Table 1 &2). In contrast, on d180, other proteins such as C8b, F2, F12, Ctsb, Ctsc, Napsin A aspartic peptidase (Napsa) and Myosin heavy chain 9 (Myh9), were down-regulated compared to controls (Supplementary Table 3).

Another common biological process, altered after exposure to TiO2 NSA in the short-term response phase, was related to cytoskeleton organization, with overexpression of various proteins such as Vimentin (Vim); Actin (Actn4, Actr3); CAP Adenylate cyclase-associated protein 1 (Cap1); Coronin 1A (Coro1A); Lymphocyte cytosolic protein 1 (Lcp1), Tubulin (Tuba3a, Tuba8, Tubb4); or downregulation of Gelsolin (Gsn) and Cofilin 2 (Cfl2) (Supplementary Table 1 & 2). While on d180, Capping actin protein of muscle Z-line alpha subunit 1 (Capza1), Gsn, Lcp1, Myosin heavy chain 9 non-muscle (Myh9) and Superoxide dismutase 1 (Sod1), were down-regulated compared to controls.

(Supplementary Table 3).

Enriched GO BP specifically modified in short-or long-term responses, are presented in Fig. 2. For instance, expression changes in the "proteasomal ubiquitin-dependent protein catabolic process" (upregulation of Psma3, Psma4, Psma6, Psmb4), cell cycle (up-regulation of Psma3-6, Psmb4, Psmb5, Psmb10, Ppp1ca-c, Tubb3), glucose metabolic process (up-regulation of Mdh2, Pgam1, Rpia, Pygl), were specific to the short-term response phase (Fig. 2A). In the long-term phase, 4 processes were specifically modified after exposure to TiO2 NSA: response to drug, regulation of blood coagulation, response to cytokine stimulus, and lipid transport (Fig. 2B).

Moreover, proteins involved in oxidation-reduction process, such as Prdx5, Lpo, Mpo, Xdh and Vat1, were mainly up-regulated in exposed rats in the short-term response phase, whereas on d180, other proteins such as Sod1, Akr1a1 and Pdia3 were down-regulated compared to controls. Interestingly, we also observed the up-regulation of various histones such as H1, H2A, H2B, H3 and H4, related to chromatin assembly, in the short-term response phase. On the other hand, 3 GO BP were enriched on day 0 and day 180 : "response to organic substance", "response to hormone stimulus" and "cytoskeleton organization". On day 3, some proteins involved in these GO BP were still differentially expressed, but these processes were not statistically enriched (p > 0.05) or didn't belong to annotation clusters with an Enrichment Score (Z score) greater than 1.3 [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF]. On d0, KEGG biological pathways were significantly enriched (p < 0.05); on d3 and d180; there were only 3 (Table 2). Like for GO BPs, KEGG pathways affected by inhalation of TiO2 NSA in the short-term response phase, were mainly related to proteolysis (proteasome pathway), inflammation (complement and coagulation cascade pathway), and cytoskeleton organization (regulation of actin cytoskeleton; gap junction pathways including members of the alpha and beta tubulin protein family).

On d180, complement and coagulation cascade pathway was still enriched, but the proteins involved were down-regulated in exposed rats compared to control rats.

To better understand the interactions between the differentially regulated proteins in response to TiO2 NSA exposure, the protein-protein networks were visualized on d0, d3 and d180 using STRING database v10.5 (Fig. 3, 4 and5). On d0, 5 major networks were identified, related to the proteasome (key node proteins Psma3, Psma4, Psma5, Psma6, Psmb4, Psmb5, Psmb10), complement and coagulation cascades (key node proteins C3, C4a, C4bpa, Plg, Serpina1), glutathione metabolism (key node proteins Gpx1, Gss, Gstm2, Gstm4, Lap3), regulation of actin cytoskeleton (key node proteins Actn4, Arpc4, Arpc5, Cfl2, Gsn, Ppp1ca, Ppp1cc, Ppp1cb), and gap junction pathways (key node proteins Tuba3a, Tuba4a, Tuba8, Tubb3) (Fig. 3). We also observed a network including various histones (H1, H2 and H3) as mentioned above. Interestingly, these 6 networks interact directly with each other, or indirectly through other proteins. On d3, 2 major networks were identified, related to the proteasome (key node proteins Psma3, Psma4, Psma6, Psma8, Psmb4), and complement and coagulation cascade pathways (key node proteins C1s, C4a, C4bpa) (Fig. 4). These 2 networks were however independent, without direct or indirect interactions.

On d180, only one major network was observed and was related to the complement and coagulation cascade pathway (key node proteins F2, F12, Kng1, Serpinc1) (Fig. 5). Some brief statistics of the inferred network, such as the number of nodes and edges are presented in Table 3. It's worth noticing that small PPI enrichment p-values were observed on d0, d3 and d180 which indicated that the nodes were not random and that the observed number of edges was significant. On d0, d3 and d180, networks had significantly more interactions than expected. This means that proteins had more interactions among themselves than what would be expected from a random set of proteins of similar size, randomly drawn from the genome. Such enrichment indicates that the proteins, as a group, were at least partially biologically connected.

Proteome profiler TM experiment

The proteome profiler experiment indicated that 9 out of the 29 cytokines analyzed, were significantly differentially expressed in BALF on d0 (p < 0.05); 8 of these proteins had increased levels including Cinc-1 (Cxcl1), Cinc-2α/β (Cxcl3), Fractalkine (Cx3cl1), Lix (Cxcl5), and only one, Thymus chemokine 1 (Cxcl7) level was decreased in exposed rats (Fig. 6A). On d3, 9 cytokines were significantly up-regulated, out of which 6 were already overexpressed on d0: Cinc-1 (Cxcl1), Cinc-2α/β (Cxcl3), Lix (Cxcl5), L-Selectin, Timp-1 and Vegf. Thymus chemokine 1 (Cxcl7), which was down-regulated on d0, was up-regulated on d3 (Fig. 6B). On d180, 4 proteins were significantly down-regulated compared to controls (p < 0.05): Cinc-2α/β (Cxcl3), Fractalkine (Cx3cl1), Lix (Cxcl5) and Vegf, whereas as mentioned above, they were up-regulated at shorter post-exposure time points (Fig. 6C).

Discussion

Exposure to TiO2 NSA

For this study, rats were exposed 6 hours per day, 5 days a week for 4 weeks to TiO2 P25 NSA at a concentration of 10 mg/m 3 . For such an aerosol (with a mass median aerodynamic diameter around 900 nm), the pulmonary deposited fraction in rat measured by ICP-MS in a previous study was around 21% of the inhaled dose [20]. In human, the estimated pulmonary deposited fraction is around 11%, according to Multiple-Path Particle Dosimetry Model (MPPD v3.04) (https://www.ara.com/products/multiple-path-particle-dosimetry-model-mppd-v-304) using asymmetric PNNL human airway morphometries [START_REF] Anjilvel | A multiple-path model of particle deposition in the rat lung[END_REF][START_REF] Miller | Improvements and additions to the Multiple Path Particle Dosimetry model[END_REF]. Taking also into account the interspecies differences in terms of respiratory minute volumes, 0.23 L/min for a Fischer 344 rat of 280 g [20] and 7.5 L/min for a man at rest (default values used by MPPD model v3.04), and total alveolar epithelial cell surface area of 0.4 m² for rat and 102 m² for human, the cumulated deposited dose normalized to alveolar cell surface area following animal exposure would be similar to the one following daily occupational exposure of a worker at 0.3 mg/m 3 (the NIOSH recommended exposure limit for ultrafine TiO2 [START_REF]Occupational Exposure to Titanium Dioxide Current Intelligence[END_REF]) throughout his career : 35 hours per week, 45 weeks per year for 20 years).

Short-term response

The strong pulmonary inflammatory response observed in the short-term response phase, was characterized by an increase in BALF total cells and neutrophils. Most studies focusing on the pulmonary response to inhaled/instilled TiO2 nanostructured particles in rats or mice [START_REF] Bermudez | Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles[END_REF][START_REF] Bermudez | Long-term pulmonary responses of three laboratory rodent species to subchronic inhalation of pigmentary titanium dioxide particles[END_REF], highlighted lung inflammation characterized by an increase in BALF total cell and neutrophil counts, total protein content and enzyme activities [START_REF] Bermudez | Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles[END_REF][START_REF] Rahman | Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses[END_REF][START_REF] Halappanavar | Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study[END_REF][START_REF] Ma-Hock | Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance[END_REF][START_REF] Nurkiewicz | Systemic microvascular dysfunction and inflammation after pulmonary particulate matter exposure[END_REF][START_REF] Warheit | Pulmonary toxicity study in rats with three forms of ultrafine-TiO2 particles: differential responses related to surface properties[END_REF].

As shown previously, animal exposure to TiO2 nanostructured particles also lead to an increase of BALF proteins probably associated with an impairment of the barrier between the alveolar wall and the blood capillaries leading to plasma proteins exudation in the alveolar space [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF]. This process may be responsible for the significant increase of some proteins from the plasmatic origin, in the BALF of exposed animals. Proteomics analysis of BALF supernatant also revealed up-regulation of proteins associated with inflammation and immune response in the short-term phase, including protein S100a8, Ctss, Mpo, Cd177, Bpifb1, and a group of complement components: C4a, C4b, Cfb and C4bpa. To complete this analysis, an antibody array experiment performed on BALF supernatant confirmed the overexpression of numerous inflammatory cytokines, including Cxcl1, Cxcl3, Cxcl5. Moreover, deregulation of proteins involved in proteasome complex (such as Psma3, Psma4, Psma6, Psmb4); glucose metabolism (such as Pgam1, Pygl, Rpia, Eno2); chromatin assembly (up-regulation of various histones on d0 and d3); cytoskeleton organization (such as Vimentin, Actin, tubulin and Cofilin); oxidative stress (Lpo, Mpo, Prdx5, Mdh) and lipid metabolism (Pla2g1b, Sftpb, Plbd1); were observed. Taken together, these data are in agreement with in vitro and in vivo proteomics studies on TiO2 NPs showing deregulation of proteins involved in these various biological processes. They were found in A549 epithelial alveolar cells chronically exposed for 2 months to 2.5 or 50 μg / mL of TiO2-NPs [START_REF] Armand | Molecular responses of alveolar epithelial A549 cells to chronic exposure to titanium dioxide nanoparticles: A proteomic view[END_REF], or exposed for 24h to 0, 60, 140 and 200 μg / cm 2 of TiO2 (SRM-154b) particles [START_REF] Vuong | Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures[END_REF] and in human bronchial epithelial cell line (BEAS-2B) exposed for 24h to BSA coated TiO2 particles [START_REF] Cha | Proteomic identification of macrophage migration-inhibitory factor upon exposure to TiO2 particles[END_REF], or exposed to 10 or 100 μg/mL of P25 TiO2 nanoparticles [START_REF] Ge | Proteome profiling reveals potential toxicity and detoxification pathways following exposure of BEAS-2B cells to engineered nanoparticle titanium dioxide[END_REF]) ; as well as in lymph nodes of mice intradermally injected with a low dose of P25 TiO2 nanoparticles [START_REF] Gao | Proteomic analysis of early response lymph node proteins in mice treated with titanium dioxide nanoparticles[END_REF]).

The investigation of significantly enriched KEGG pathways and protein-protein interactions, revealed the presence of 7 enriched pathways and 5 major networks related to proteasome, complement and coagulation cascades, glutathione metabolism, regulation of actin cytoskeleton, and gap junction pathways. These pathways are all related to inflammation. Indeed, the ubiquitinproteasome system, the principal pathway of non-lysosomal proteolysis of intracellular proteins; is involved in the modulation of the immune and inflammatory responses [START_REF] Wang | The ubiquitin-proteasome system and its role in inflammatory and autoimmune diseases[END_REF]. Furthermore, glutathione metabolism participates in antioxidant defense systems that are involved in response to oxidative stress, induced by inflammation [START_REF] Khansari | Chronic inflammation and oxidative stress as a major cause of age-related diseases and cancer[END_REF][START_REF] Rogers | The cytoskeleton and its importance as a mediator of inflammation[END_REF].

During an inflammatory response, the recruitment and activation of neutrophils can cause oxidative stress. Here, we observed that the levels of some BALF proteins involved in this process were elevated following exposure to TiO2 NSA. Indeed, mass spectrometry analysis revealed increased levels of Lpo, Mpo, and anti-oxidant proteins Hspa1a, Hspa1b, Prdx5 in the short-term response phase. The latters are involved in ROS scavenging systems, suggesting that defense mechanisms against oxidative stress were triggered. However, despite their antioxidant activities, proteins from the superoxide dismutase family were not differentially expressed in the short-term response phase. As a whole our results are in good agreement with previous in vivo and in vitro studies indicated that TiO2 NPs deposited into the lung trigger ROS production [START_REF] Shi | Titanium dioxide nanoparticles: a review of current toxicological data[END_REF].

In addition enriched biological pathways found after TiO2 exposure such as oxidative stress, proteolysis, inflammatory response and cytoskeleton organization share similarities with those identified in chronic obstructive pulmonary disease (COPD) patients and smokers [START_REF] Rollet-Cohen | Comparative proteomics of respiratory exosomes in cystic fibrosis, primary ciliary dyskinesia and asthma[END_REF][START_REF] Yang | Long-term smoking alters abundance of over half of the proteome in bronchoalveolar lavage cell in smokers with normal spirometry, with effects on molecular pathways associated with COPD[END_REF][START_REF] Yang | Proteomic profiling of lung immune cells reveals dysregulation of phagocytotic pathways in female-dominated molecular COPD phenotype[END_REF]. These results may suggest that a sustained exposure to TiO2 NP level high enough to trigger chronic inflammation may lead to the development of pulmonary pathologies.

Interestingly, mass spectrometry-based proteomics analysis of BALF showed increased levels of a number of histone isoforms on d0 and d3: Histones H1.4, H2A, H2B and H4. Histones are intracellular proteins involved in nucleosome assembly. However, these proteins can also be released in the extracellular space by neutrophils during the neutrophil extracellular traps (NET) formation [START_REF] Daigo | The Protective Effect against Extracellular Histones Afforded by Long-Pentraxin PTX3 as a Regulator of NETs[END_REF], or through neutrophil-mediated cell damage and death [START_REF] Ward | Acute lung injury and the role of histones[END_REF][START_REF] Urban | Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans[END_REF]. Indeed, neutrophils are recruited to the site of injury, to recognize and phagocyte pathogens through various cytotoxic mechanisms; such as ROS production, release of antimicrobial peptides and expulsion of their nuclear contents to form NET [START_REF] Mayadas | The multifaceted functions of neutrophils[END_REF]. NET formation or NETosis is considered to be the main source of extracellular histones [START_REF] Daigo | The Protective Effect against Extracellular Histones Afforded by Long-Pentraxin PTX3 as a Regulator of NETs[END_REF].

NET are composed of decondensed chromatin fibers coated with antimicrobial proteins such as neutrophil Elastase, Myeloperoxidase (Mpo) or Lysozyme. In 2009, a proteomic analysis identified 24 NET-associated proteins in human peripheral blood neutrophils including histones [START_REF] Urban | Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans[END_REF]. In our study, 7 out of these 24 proteins were found in BALF supernatant of exposed rats: histones H2A, H2B, H4, Mpo, Lysozyme, S100a8 and alpha-actinin 4 [START_REF] Daigo | The Protective Effect against Extracellular Histones Afforded by Long-Pentraxin PTX3 as a Regulator of NETs[END_REF][START_REF] Ward | Acute lung injury and the role of histones[END_REF][START_REF] Urban | Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans[END_REF]. This might be explained by the sensitivity of our method or the way samples were collected which did not allow identifying all the differentially expressed extracellular proteins present in the BALF especially those with low abundance.

NET release induced by nanoparticles was described in vitro with human primary immune cells exposed to gold nanoparticles [START_REF] Bartneck | Phagocytosis independent extracellular nanoparticle clearance by human immune cells[END_REF]. Another study reported disturbance in NETosis and oxidative burst induced by TiO2 NPs [START_REF] Jovanovic | Effects of nanosized titanium dioxide on innate immune system of fathead minnow (Pimephales promelas Rafinesque, 1820)[END_REF] both in vivo on fathead minnow fish (Pimpehales promelas), and in vitro using cell suspension obtained from adult fish kidneys. Recently, it has been suggested that 15-nm silver NPs also induce NET release from human polymorphonuclear neutrophils isolated from venous blood of healthy volunteers [START_REF] Liz | Silver nanoparticles rapidly induce atypical human neutrophil cell death by a process involving inflammatory caspases and reactive oxygen species and induce neutrophil extracellular traps release upon cell adhesion[END_REF].

Although NET formation is involved in neutrophil host defense functions, excessive NET release can cause tissue damage, stimulation of undesirable immune reactions, and is associated with various pulmonary diseases such as acute lung injury, asthma, cystic fibrosis, chronic obstructive pulmonary disease [START_REF] Mayadas | The multifaceted functions of neutrophils[END_REF][START_REF] Porto | Neutrophil Extracellular Traps in Pulmonary Diseases: Too Much of a Good Thing?[END_REF]. NET associated proteins were also found in BALF from Calves infected by bovine respiratory syncytial virus [START_REF] Hagglund | Proteome analysis of bronchoalveolar lavage from calves infected with bovine respiratory syncytial virus-Insights in pathogenesis and perspectives for new treatments[END_REF] suggesting that pulmonary mechanisms involved in the defense against foreign nanomaterials share common pathways with those involved with viruses. Here, this possible NET release occurs in an inflammatory context but in the absence of significant histopathological changes. Indeed, as previously described, neither epithelial cell apoptosis/necrosis in lung epithelial cells, nor fibrosis in the airways was observed [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF]. In addition, NET release was only observed in the acute phase suggesting that it was associated the strong inflammatory response which disappears overtime.

Further experiments are required to better characterize the source of extracellular histones in BALF such as immunostaining which can visualize the co-localization of chromatin and granule proteins such as Mpo [START_REF] Mayadas | The multifaceted functions of neutrophils[END_REF]. Nevertheless, taken together, these findings could be linked to the stimulation of NET formation in rat lung following nose-only exposure to TiO2 NSA which, to the best of our knowledge, has not been described before.

Long-term response

Activation of defense mechanisms immediately after the exposure to nanomaterials is a physiological process. However, the long-term consequences of this type of exposure raise concerns since it may lead to various pulmonary pathologies [START_REF] Shi | Titanium dioxide nanoparticles: a review of current toxicological data[END_REF][START_REF] Song | Contribution of oxidative stress to TiO2 nanoparticleinduced toxicity[END_REF]. The amount of titanium (measured by ICP-MS) found in the lung of the exposed animals at d0 was about 2.1 mg/lung [20]. It decreased overtime and reached 35% of the starting amount on d180. This results suggest that a significant amount of particles were still present in the lung 180 days after the end of exposure but had limited physiopathological effects. An inflammatory response was still present, however, at a much lower intensity than in the short-term response. It was associated with the presence of particles-laden macrophages. But, as previously described, despite the presence of remaining nanoparticles in the lung, no significant histopathological change was observed [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF]. On d180, most of the proteins with altered expression in BALF supernatant, as assessed by mass spectrometry, were down-regulated compared to controls, except for Trem2 (Triggering receptor expressed on myeloid cells 2), Cd177 antigen-like and an uncharacterized protein involved in inflammation. Three enriched pathways and one major interaction network (complement and coagulation cascades pathway), were still observed, related to the persistence of a low intensity inflammatory response. Additionally, a slight but significant down-regulation of Vegf and 3 inflammatory chemokines (Cxcl3, C3xcl1, Cxcl5) was detected in BALF. Vegf is secreted by alveolar macrophages to initiate repair of the endothelial barrier following lung inflammation; Cxcl3 and Cxcl5 are involved in the recruitment and activation of neutrophils to alveolar spaces [START_REF] Herold | Acute lung injury: how macrophages orchestrate resolution of inflammation and tissue repair[END_REF][START_REF] Aggarwal | Diverse macrophage populations mediate acute lung inflammation and resolution[END_REF]. Down-regulation of some chemokines in exposed rats on d180 might be related to the decrease in BALF neutrophils in long term response, supporting a progressive resolution of inflammation and potential lung recovery [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF], related to the decrease of the Ti lung burden [20]. These data are consistent with the fact that TiO2 nanoparticles are low toxicity, low solubility nanomaterials and clearly show that defense mechanisms initiated during the exposure to aerosol were effective to circumvent any longterm adverse effects that could been induced by these nanomaterial despite the relatively high initial pulmonary deposited dose.

During the inflammatory response, excessive ROS production leads to the development of oxidative stress. In BALF, some proteins involved in oxidative stress (Hspa1a, Hspa1b and Sod1) were down-regulated on d180 in exposed rats compared to control rats. As mentioned above, these results suggest a progressive resolution of oxidative stress in the long-term response phase associated with the presence of few remaining inflammatory cells.

Comparison of proteomic and transcriptomic data in short-and long-term responses

A previous study using transcriptomic analysis was performed on rat lungs [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF]. Comparing these results with our present proteomics work, we noticed a common overexpression transcripts and proteins from genes involved in inflammatory and immune responses, complement and coagulation cascades, and oxidative stress. Among those, chemokines (Cxcl1, C3xcl1, Cxcl5), and others like Cd177, C4bpa, Lpo, Chia and Tuba8 were identified [START_REF] Chezeau | Short-and long-term gene expression profiles induced by inhaled TiO2 nanostructured aerosol in rat lung[END_REF]. Six months after the end of exposure, despite the fact that the number of inflammatory cells had decreased; many genes and proteins remained differentially expressed. These data tend to show a link between transcriptomic and proteomic data suggesting that the change in the expression of some genes in the lung leads to the secretion of proteins in the alveolar space where they could play a role in the defense mechanisms activated following TiO2 exposure.

Conclusion

This in vivo study brings new insight about the short-and long-term pulmonary effects (up to 6month post-exposure) of inhaled nanostructured TiO2 aerosol through the combination of BALF cytology and proteomics analyses. Exposure to TiO2 NSA resulted in a strong acute inflammatory response in the lung. This response was characterized by a neutrophil influx, associated with an overexpression of proteins involved in proteasome, inflammatory and immune responses, complement and coagulation cascade, oxidative stress, and cytoskeleton organization in the short-term response phase. In the BALF supernatant, the increased level of histones and other NET-associated proteins suggests the release of NET in the alveolar space. This possible NET release occurs in an inflammatory context but in the absence of significant histopathological changes.

Six months after the end of exposure (long-term response), inflammation had decreased, but many proteins remained differentially expressed; mainly down-regulated in exposed rats compared to control rats. These observations suggest a progressive resolution of inflammation and potential lung recovery.

We observed changes in processes such as inflammation and cardiovascular system. These 2 processes are linked especially via Kng1, also known as HMWK-kallikrein factor, which expression was altered on d180. Thus, Kng1 might be a good candidate biomarker for monitoring the pulmonary toxicity induced by nanomaterials, although this hypothesis needs to be further investigated. enriched Gene Ontology Biological Processes (GO BP) modified in BALF of rats exposed to TiO2 nanostructured aerosol immediately, 3 and 180 days after the end of exposure. They were determined through DAVID bioinformatics Resources 6.7 with a Z score > 1.3 or a pvalue < 0.05. Fig. 2 Enriched GO BP specifically modified in the short-(A) or long-term (B) response phase in BALF of rats exposed to TiO2 nanostructured aerosol. (Z score > 1.3 or p < 0.05). Fig. 3 The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, immediately after the end of exposure. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, immediately after the end of exposure (p < 0.05) (Supplementary Table 1) were injected in STRING database v10.5. Some significantly enriched KEGG pathways (determined using DAVID database) are represented in the boxes. Legend: Network nodes represent proteins, edges represent protein-protein associations (red line: presence of fusion evidence, green line: neighborhood evidence, blue line: co-occurrence evidence, purple line: experimental evidence, yellow line: text mining evidence, light blue line: database evidence, black line: co-expression evidence). Fig. 4 The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 3. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 3 (p < 0.05) (Supplementary Table 2) were injected in STRING database v10.5. Some significantly enriched KEGG pathways (determined using DAVID database) are represented in the boxes. Fig. 5 The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 180. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 180 (p < 0.05) (Supplementary Table 3) were injected in STRING database v10.5. One significantly enriched KEGG pathway (determined using DAVID database) is represented in the boxes. Fig. 6 Significantly expressed cytokines in BALF of exposed animals. Expression level of 29 cytokines was analyzed in control and exposed rats to TiO2 nanostructured aerosol in BALF on post-exposure days 0, 3 and 180. Only differentially expressed proteins (p < 0.05) are presented.

Figures Fig. 1

 1 FiguresFig.1Common enriched Gene Ontology Biological Processes (GO BP) modified in BALF of rats exposed to TiO2 nanostructured aerosol immediately, 3 and 180 days after the end of exposure. They were determined through DAVID bioinformatics Resources 6.7 with a Z score > 1.3 or a pvalue < 0.05. Fig.2Enriched GO BP specifically modified in the short-(A) or long-term (B) response phase in BALF of rats exposed to TiO2 nanostructured aerosol. (Z score > 1.3 or p < 0.05). Fig.3The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, immediately after the end of exposure. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, immediately after the end of exposure (p < 0.05) (Supplementary Table1) were injected in STRING database v10.5. Some significantly enriched KEGG pathways (determined using DAVID database) are represented in the boxes. Legend: Network nodes represent proteins, edges represent protein-protein associations (red line: presence of fusion evidence, green line: neighborhood evidence, blue line: co-occurrence evidence, purple line: experimental evidence, yellow line: text mining evidence, light blue line: database evidence, black line: co-expression evidence). Fig.4The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 3. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 3 (p < 0.05) (Supplementary Table2) were injected in STRING database v10.5. Some significantly enriched KEGG pathways (determined using DAVID database) are represented in the boxes. Fig.5The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 180. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 180 (p < 0.05) (Supplementary Table3) were injected in STRING database v10.5. One significantly enriched KEGG pathway (determined using DAVID database) is represented in the boxes. Fig.6Significantly expressed cytokines in BALF of exposed animals. Expression level of 29 cytokines was analyzed in control and exposed rats to TiO2 nanostructured aerosol in BALF on post-exposure days 0, 3 and 180. Only differentially expressed proteins (p < 0.05) are presented.

  FiguresFig.1Common enriched Gene Ontology Biological Processes (GO BP) modified in BALF of rats exposed to TiO2 nanostructured aerosol immediately, 3 and 180 days after the end of exposure. They were determined through DAVID bioinformatics Resources 6.7 with a Z score > 1.3 or a pvalue < 0.05. Fig.2Enriched GO BP specifically modified in the short-(A) or long-term (B) response phase in BALF of rats exposed to TiO2 nanostructured aerosol. (Z score > 1.3 or p < 0.05). Fig.3The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, immediately after the end of exposure. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, immediately after the end of exposure (p < 0.05) (Supplementary Table1) were injected in STRING database v10.5. Some significantly enriched KEGG pathways (determined using DAVID database) are represented in the boxes. Legend: Network nodes represent proteins, edges represent protein-protein associations (red line: presence of fusion evidence, green line: neighborhood evidence, blue line: co-occurrence evidence, purple line: experimental evidence, yellow line: text mining evidence, light blue line: database evidence, black line: co-expression evidence). Fig.4The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 3. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 3 (p < 0.05) (Supplementary Table2) were injected in STRING database v10.5. Some significantly enriched KEGG pathways (determined using DAVID database) are represented in the boxes. Fig.5The STRING network of protein-protein interactions in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 180. List of all differentially regulated proteins in BALF of rats exposed to TiO2 nanostructured aerosol, on post-exposure day 180 (p < 0.05) (Supplementary Table3) were injected in STRING database v10.5. One significantly enriched KEGG pathway (determined using DAVID database) is represented in the boxes. Fig.6Significantly expressed cytokines in BALF of exposed animals. Expression level of 29 cytokines was analyzed in control and exposed rats to TiO2 nanostructured aerosol in BALF on post-exposure days 0, 3 and 180. Only differentially expressed proteins (p < 0.05) are presented.

Table 1 :

 1 Aerosol characterisation.

	1
	2
	Particle size Specific surface area	Aerosol mass concentration	Aerosol number concentration	CMAD (nm) MMAD (nm)
	(nm)	(m 2 /g)	(mg/m 3 )	(particle/cm 3 )
	21.5 ± 7.2	51	10.17 ± 3.29	24000 ± 6400	269	905
					(GSD : 2.22) (GSD : 2.19)
	3
	4

Table 2 :

 2 Significantly enriched biological pathways in BALF of rats exposed to TiO2 nanostructured

	5
	6	aerosol, on post-exposure days 0, 3 and 180 (p-value < 0.05).
	7
		KEGG Pathways	Number of genes	p-value	Proteins
		Day 0
		Proteasome	8	3.3447E-7	PSMB10, PSMB5, PSMB4, PSMA6, PSMA5, PSMA4, PSMA3, RGD1564425
		Complement and coagulation cascades	6	4.7987E-4 CFB, C4B, C3, SERPINA1, C4BPA, PLG
		Glutathione metabolism 5	0.0012	GSS, LAP3, GPX1, GSTM2, GSTM4
		Systemic lupus erythematosus	6	0.0015	ACTN4, HIST1H4B, C4B, C3, HIST2H2BE, HIST3H2BA
		Regulation of actin cytoskeleton	8	0.0032	PPP1CA, ACTN4, GSN, CFL2, ARPC4, ARPC5, PPP1CC, PPP1CB
		Oocyte meiosis	5	0.0204	PPP1CA, PPP2CA, PPP2CB, PPP1CC, PPP1CB
		Gap junction	4	0.0408	TUBA8, TUBA3A, TUBA4A, TUBB3
		Day 3
		Proteasome	4	3.4747E-4 PSMB4, PSMA6, PSMA4, PSMA3
		Complement and coagulation cascades	4	9.3708E-4 CFB, C4B, C1S, C4BPA
		Systemic lupus erythematosus	3	0.0265	HIST1H4B, C4B, C1S
		Day 180
		Complement and coagulation cascades	6	2.7045E-6 KNG1, C8B, F12, C4B, F2, SERPINC1
		Prion diseases	3	0.0060	C8B, HSPA1A, HSPA1B, SOD1
		Antigen processing and presentation	3	0.0352	PDIA3, HSPA1A, CTSB, HSPA1B
	8
	9

Table 3 :

 3 Brief statistics of the inferred network calculated on post-exposure days 0, 3 and 180 (String 10 database V10.5).

	11
	12
		Post-exposure days
		0	3	180
	Number of nodes	101	43	40
	Number of edges	204	26	19
	Average node degree	4.04	1.21	0.95
	Average local clustering coefficient	0.526	0.326	0.395
	Expected number of edges	73	4	6
	PPI enrichment p-value	< 1.0e-16	1.17e-12	1.57e-05
	13

Acknowledgements

This work has been supported by the National Fund for the Prevention of Occupational Accidents and Diseases.

The authors gratefully acknowledge Aurélie Remy for her help with statistical analysis, Stéphane

Grossmann and Hervé Nunge for aerosol generation and characterization, Sylvie Sébillaud, Mylène Lorcin and Cristina Langlais for tissue sampling and BAL cell count, and Sylvie Michaux, Marie-Josèphe Décret and Laurine Douteau for animal care and husbandry. Nathalie Quintero is acknowledged for her help with the mass spectrometry work performed at the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory (UC Berkeley, CA).

Conflicts of interest

The authors declare that they have no competing interests.


Guillaume Laconde

Muriel Amblard

Jean Martinez

Synthesis of α-Amino Acid N-Carboxyanhydrides

ABSTRACT: A simple phosgene-and halogen-free method for synthesizing α-amino acid N-carboxyanhydrides (NCAs) is described. The reaction between Boc-protected α-amino acids and T3P ® reagent gave the corresponding NCA derivatives in good yield and purity with no detectable epimerization. The process is safe, easy-to-operate and does not require any specific installation. It generates non-toxic, easy to remove by-products. It can apply to the preparation of NCAs for the on-demand on-site production of either little or large quantities. α-Amino acid-N-carboxyanhydrides (NCAs) (oxazolidine-2,5dione derivatives) are cyclic activated amino acid building blocks that are highly reactive with nucleophiles, [START_REF] Bouifraden | Some of the Amino Acid Chemistry Going on in The Laboratory of Amino Acids, Peptides and Proteins[END_REF] producing only carbon dioxide as a by-product. This feature has highlighted their usefulness in creating amide bonds. [START_REF] Kricheldorf | α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles: Syntheses, Properties, Peptide Synthesis, Polymerization[END_REF] NCAs are also the main reagents used in the synthesis of well-defined peptide polymers 3a-c that have recently grown in importance and are now used in some of the most innovative healthcare strategies [START_REF] Duro-Castano | Peptide-Based Polymer Therapeutics[END_REF] including controlled drug/gene delivery systems, tissue engineering, molecular imaging, and diagnostics. [START_REF] Nitta | Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering[END_REF] As an example, Glatimer, which is prepared through the random copolymerization of (L)Ala-NCA, (L)Glu(OBzl)-NCA, (L)Lys(Tfa)-NCA, and (L)Tyr-NCA, is commercially available for the treatment of multiple sclerosis. [START_REF] Bell | Development of Glatopa® (Glatiramer Acetate): The First FDA-Approved Generic Disease-Modifying Therapy for Relapsing Forms of Multiple Sclerosis[END_REF] NCAs have also been used to produce several active pharmaceutical ingredients e.g., the antiviral drug Saquinavir and Nelfinavir intermediates, [START_REF] Robin | α-Amino Acid N-Carboxy Anhydrides in Pharmaceutical Innovations: Try Them to Capture New Value[END_REF] and polypeptide-based polymer therapeutics that are currently in clinical trials including the poly(glutamic acid)paclitaxel conjugate Opaxio TM for treating various cancers and the poly(ethylene glycol) methyl ether-poly(aspartic acid)paclitaxel micelle NK-105 for treating breast cancer. [START_REF] Hamaguchi | NK105, a Paclitaxel-Incorporating Micellar Nanoparticle Formulation, Can Extend in Vivo Antitumour Activity and Reduce the Neurotoxicity of Paclitaxel[END_REF] Poly(glutamic acid) is predicted to become an important biopolymer, [START_REF] Park | Poly(glutamic acid): Production, composites, and medical applications of the next-generation biopolymer[END_REF] and poly(lysine) and poly(aspartic acid) have been developed for use as carriers, because they have high drug loading capacities and are biodegradable. [START_REF] Johnson | Recent developments in polymer-block-polypeptide and protein-polymer bioconjugate hybrid materials[END_REF] On the other hand, (L)Leu-NCA is produced industrially for the synthesis of polyleucine, which is used as a catalyst in the Julia-Colonna asymmetric epoxidation method. 11a-b NCAs are also useful intermediates for the "green" solvent-free synthesis of peptides. [START_REF] Declerck | Solvent-Free Synthesis of Peptides[END_REF] Leuchs was the first to develop a method for synthesizing αamino acid NCAs (Leuchs's anhydrides) by treating Nalkyloxycarbonyl amino acids with halogenating agents (e.g. PBr 3 , PCl 3 , PCl 5 , SOCl 2 , α,α-dichloromethylmethyl ether), which result in the formation of carcinogenic alkyl halides; 13a-c other methods have since been developed. These include the reaction of phosgene with α-amino acids (the Fuchs-Farthing method), 14a-b and methods involving a nongaseous phosgene substitute (diphosgene or triphosgene). 15a-b A method for synthesizing NCAs using phosgene through basic-to-acidic flash switching in a microflow reactor has recently been described, and allows the synthesis of NCAs bearing acid-labile protecting groups. [START_REF] Otake | Rapid and Mild Synthesis of Amino Acid N -Carboxy Anhydrides: Basic-to-Acidic Flash Switching in a Microflow Reactor[END_REF] So far, the most common methods for preparing NCAs involve phosgene, but the high toxicity of phosgene and legal restrictions on its use, which can cause the release of corrosive gaseous HCl, mean that such methods are difficult to start up. Some phosgene-free methods for synthesizing NCAs have been developed to avoid these drawbacks. Some of these methods have involved the use of NO gas and oxalyl chloride, 17a-b di-tert-butyltricarbonate, [START_REF] Kudo | Synthesis and Self-Polyaddition of Optically Active Monomers Derived from Tyrosine[END_REF] bisaryl carbonates, 19a-b and cyanuric chloride. [START_REF] Enomoto | Synthesis of Peptide-Grafted Comb Polypeptides via polymerisation of NCA-Peptides[END_REF] Unfortunately, none of these have become popular due to their incompatibility with some protecting groups, the formation of side products, the toxicity of the reactants, and complications due to the large number of steps in the reaction. The Fuchs-Farthing method is still the most widely used for synthesizing NCAs, particularly in industrial settings. There is therefore a clear need for a mild, non-acidic, safe, easy-to-use process for producing a wide range of NCAs in good yields, high purities, based on reagents that are chemically compatible and apparatus that is widely available, and which produces inoffensive by-products that can be removed easily. n-Propanephosphonic acid anhydride (T3P ® ) has been found to be useful for activating carboxylic acids and forming amide bonds under mild conditions. 21 T3P ® is a non-toxic reagent that does not require specific handling measures. It is one of the most widely used carboxylic acid activating reagents for large-scale syntheses. 22a-b We recently reported an interesting property of T3P ® in that it can activate carboxylic acids to yield simply and cleanly αamino acid N-acyl-benzotriazole derivatives [START_REF] Laconde | A Simple and Versatile Method to Synthesize N-Acyl-Benzotriazoles[END_REF] that have unexpected reactivities towards weak nucleophiles in acidic media. [START_REF] Laconde | Unexpected Reactivity of N-Acyl-Benzotriazoles with Aromatic Amines in Acidic Medium (ABAA Reaction)[END_REF] From our study of the synthesis of N-acyl-benzotriazole derivatives, when synthesizing Boc-(L)Tyr(OtBu)-OBt from Boc-(L)Tyr(OtBu)-OH in the presence of T3P ® , pyridine and benzotriazole, in AcOEt as solvent, we noticed faint traces of a side product that was identified unexpectedly as (L)Tyr(OtBu)-NCA. We then performed the reaction using Boc-(L)Tyr(OtBu)-OH, 1.2 T3P ® eq in AcOEt as solvent, under the same experimental conditions, but with no benzotriazole. Surprisingly, some (L)Tyr(OtBu)-NCA formed rapidly after a 2 h reaction (Scheme 1).

Scheme 1. Synthesis of NCAs

We decided to study this interesting reaction further. We first explored the effect of temperature on the reaction. By increasing the reaction temperature to 50 °C it was possible to achieve a high Boc-(L)Tyr(OtBu)-OH conversion rate, and (L)Tyr(OtBu)-NCA was isolated with a good yield. We then investigated the use of a base (2 eq) as a "reaction facilitator", including pyridine, 4-dimethylamino-pyridine, Nmethylmorpholine, N,N-diisopropyl ethylamine, triethylamine, 1,8-diazabicyclo [5.4.0]undec-7-ene, and the inorganic base K 2 CO 3 . Each reaction was performed at room temperature for 2 h using EtOAc as the solvent. (L)Tyr(OtBu)-NCA was formed using all the tertiary amines, but the conversion rate in most cases was low, and side products were formed. K 2 CO 3 caused almost no conversion to occur. Pyridine was the most effective base, giving the highest conversion rate, the cleanest crude product, and the best NCA yield. These results prompted us to evaluate the role of pyridine as a "catalyst" in more detail. We synthesized (L)Tyr(OtBu)-NCA from Boc-(L)Tyr(OtBu)-OH in the presence of different amounts of pyridine (0.25-2 eq) using AcOEt as a solvent and a 18 h reaction time. Although formation of the NCA was observed using 0.25 and 0.5 pyridine equivalents, the best conversion and recovery of (L)Tyr(OtBu)-NCA was obtained when 1 or 2 equivalents of pyridine were used (Figure 1SI). In addition, it appeared that 1.2 T3P ® equivalent was the best amount to obtain a clean and total conversion (Figure 2SI). It has been reported that activation of a carboxylic acid by T3P ® followed by formation of an amide bond in the presence of a nucleophilic amine does not induce epimerization. [START_REF] Dunetz | General and Scalable Amide Bond Formation with Epimerization-Prone Substrates Using T3P ® and Pyridine[END_REF] However, we investigated whether epimerization occurred during the synthesis of the NCA derivatives in the presence of pyridine. We reacted Boc-(L)Asp(OBzl)-OH 1 (1 eq) and Boc(D)Asp(OBzl)-OH 2 (1 eq) with T3P ® (1.2 eq) and pyridine (1 eq) in EtOAc at room temperature. The corresponding (L) and (D)Asp(OBzl)-NCAs 3 and 4 were obtained. They were reacted with (S)-1-(4-methoxyphenyl)-ethan-1-amine to give diastereomers 5 and 6 (Scheme 2) that were analyzed separately by HPLC, and after co-injection of a 1:1 mixture (Figures 1SI and2SI). Each compound 5 and 6 produced a single peak, indicating no detectable epimerization of the (L) and (D)Asp(OBzl)-NCAs. These results were also confirmed by 1 H NMR analysis of diastereomers 5 and 6 (SI). Scheme 2. Epimerization study of Asp(OBzl)-NCA. Synthesis of diastereomers 5 and 6.

The optimized experimental conditions were used to prepare a series of NCAs (Table 1). The NCAs included some "difficultto-prepare" NCAs bearing acid-labile protecting groups [e.g., (L)Trp(Boc)-NCA, (L)Arg(Pbf)-NCA, (L)Cys(Trt)-NCA as well as (L)Lys(Boc)-NCA for which no cyclization was observed with the N-Boc group side chain]. We also found that the method could be used to prepare the αdisubstituted amino acid NCA, i.e., Aib-NCA, N-substituted α-amino acid NCAs including N-methyl-(L)Ala-NCA, and Nbenzyl-Gly-NCA, as well as isatoic anhydride (Table 1). All the NCAs were synthesized at around the gram scale, and were obtained in good yields and at excellent purities after simple precipitation and recrystallization. The detailed physicochemical properties of the NCAs are reported in the Supporting Information. We found that the Boc protecting group and T3P ® were both necessary for the cyclization reaction. In fact, no conversion occurred when Z-(L)Phe-OH or Fmoc-(L)Phe-OH was used as the starting material and T3P ® (1.2 eq) was used, at either 50°C or using 1 eq pyridine. Neither of these phenylalanine Nprotected amino acids produced NCA derivatives. In another set of experiments, Boc-(L)Phe-OH was reacted with various carboxylic acid activating reagents instead of T3P ® . These activating reagents were N,N'-dicyclohexylcarbodiimide (2 eq), 1-[bis (dimethylamino)methylene]-1H-1,2,3-triazolo [4,5b]pyridinium-3-oxide hexafluorophosphate (2 eq), 25 and ((1Hbenzo[d][1,[START_REF] Kricheldorf | α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles: Syntheses, Properties, Peptide Synthesis, Polymerization[END_REF]3]triazol-1-yl)oxy)tris(dimethylamino) phosphonium hexafluorophosphate (2 eq). [START_REF] Castro | Réactifs de couplage peptidique I (1)-l'hexafluorophosphate de benzotriazolyl N-oxytrisdimethylamino phosphonium (B.O.P.)[END_REF] The experiments with the carboxylic acid activating reagents were performed using the same experimental conditions as those used for the experiments with T3P ® . Either a faint trace or no (L)Phe-NCA was detected by HPLC.

Scheme 3. Mechanism of NCAs formation

The results led to our proposal of the mechanism shown in Scheme 3 for the reaction. To determine whether the method could be scaled up, it was used to prepare (L)Leu-NCA from Boc-(L)Leu-OH ( 10 In conclusion, we have developed a mild, non-acidic process for synthesizing α-amino acid NCAs from N-Boc-protected α-amino acid derivatives using T3P® as an activating reagent. The process is safe and easy to perform and does not require any specialist equipment. The process generates nontoxic, easy-to-remove by-products and allows a wide range of NCAs to be synthesized with good yields and high puri-ties. The process is chemically compatible with many amino acid protecting groups that are commonly used in peptide syntheses. The method extends the range of NCA building blocks that can be used to synthesize active pharmaceutical ingredients and peptide-based therapeutic polymers. The yield does not decrease when the process is scaled up, and is easy to perform using simple benchtop equipment. The final product can be recovered easily at an excellent level of purity. The process could be used to prepare NCAs on-demand and on-site in small or large quantities using safe and simple experimental conditions.

General procedure for the preparation of -Amino Acid N-Carboxyanhydrides (NCAs)

To the Boc-AA-OH (1 eq) dissolved in AcOEt (50 ml) was added pyridine (1 eq), then T3P ® /AcOEt 50% (1.2 eq) was added dropwise. The mixture was stirred at room temperature for 2 h. The reaction was monitored by LC/MS. The mixture was then diluted in AcOEt and washed twice with cold water and then with a saturated NaCl solution. The organic layer was dried over anhydrous magnesium sulphate. The solvent was removed under reduced pressure, and the crude product crystallized. α-Amino acid Ncarboxyanhydride derivatives were obtained through simple recrystallization and were not purified further. The analytical results and physical properties of the products are shown in Table 1 and Supporting Information. Alternatively, the reaction could be performed without pyridine but at 50 °C, keeping the other conditions the same.

  g), (L)Lys(Boc)-NCA from Boc-(L)Lys(Boc)-OH (10 g), (L)Phe-NCA from Boc-(L)Phe-OH (20 g), (L)Glu(OBzl)-NCA from Boc-(L)Glu(OBzl)-OH (25 g), and (L)Ala-NCA from Boc-(L)Ala-OH (50 g). All these NCAs were obtained at good purities and in good yields [83%, 83%, 83%, 80%, and 77% for (L)Leu-NCA, (L)Lys(Boc)-NCA, (L)Phe-NCA, (L)Glu(OBzl)-NCA, and (L)Ala-NCA, respectively] after simple workup and simple recrystallization (see SI).

Table 1 . Synthesis of NCAs

 1

	NCA	Yield%*	Mp °C
	(L)Trp(Boc)-NCA	80	153-154
	(L)Arg(Pbf)-NCA	85	122-123
	(L)Val-NCA	78	62-63
	(L)Tyr(OtBu)-NCA	90	109-110
	(L)Phe-NCA	85	88-89
	(L)Asp(OBzl)-NCA	85	123-124
	(D)Asp(OBzl)-NCA	80	123-124
	(L)Thr(OBzl)-NCA	80	120-121
	(L)Gln(Xan)-NCA	78	156-157
	(L)Asn(Xan)-NCA	90	145-146
	(L)Ala-NCA	75	84-85
	(L)Ser(OBzl)-NCA	78	68-69
	(L)Glu(OBzl)-NCA	78	110-111
	(L)Cys(Trt)-NCA	80	122-123
	(L)Lys(Boc)-NCA	85	142-143
	(L)Lys(Fmoc)-NCA	88	101-102
	(L)Tic-NCA	76	166-167
	(L)Leu-NCA	86	67-69
	Aib-NCA	76	72-73
	N-Me-(L)Ala-NCA	71	131-132
	N-Benzyl-Gly-NCA	76	152-153
	Isatoic anhydride	65	242-243
	NCAs reported in this Table were prepared under the following
	experimental conditions: Boc-amino acid derivative (1 eq), T3P ®
	(1.2 eq), pyridine (1 eq) in EtOAc at r.t. for 2 h. *Crystalline pure
	compounds.